XANTHINE DEHYDROGENASE (XDH) iRNA COMPOSITIONS AND METHODS OF USE THEREOF

ABSTRACT

The present invention relates to RNAi agents, e.g., dsRNA agents, targeting the xanthine dehydrogenase (XDH) gene. The invention also relates to methods of using such RNAi agents to inhibit expression of an XDH gene and to methods of treating or preventing an XDH-associated disease in a subject.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/505,732, filed on Oct. 20, 2021, which is a 35 § U.S.C. 111(a)continuation application which claims the benefit of priority toPCT/US2021/037748, filed on Jun. 17, 2021, which—in turn—claims thebenefit of priority to U.S. Provisional Application No. 63/040,587,filed on Jun. 18, 2020, and U.S. Provisional Application No. 63/153,983,filed on Feb. 26, 2021. The entire contents of each of the foregoingapplications are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 25, 2022, isnamed 121301_12904_SL.txt and is 714,638 bytes in size.

BACKGROUND OF THE INVENTION

Reduced renal clearance of uric acid is the result of a number offactors, including defects in uric acid transporter proteins such asSLC2A9, ABCG2, and others, reduced renal excretion due to renal disease,hypothyroidism, volume contraction and volume depletion, acidosis, leadintoxication, and familial nephropathy due to uromodulin deposits; andaltered renal clearance due to hyperinsulinemia or insulin resistance indiabetes. Increased synthesis of uric acid is associated withhyperuricemia plus hyperuricosuria; inborn errors of metabolism such asLesch Nyhan/HPRT deficiency, PRPP synthetase overactivity, andglucose-6-phosphate dehydrogenase deficiency (Von Gierkedisease/Glycogen Storage Disease Type Ia); certain situations of highcell turnover (e.g., tumor lysis syndrome); certain situations of highATP turnover (e.g., glycogen storage diseases, tissue ischemia).Furthermore, conditions such as chronic kidney disease, hypertension,metabolic syndrome, and high fructose intake may result in bothincreased uric acid synthesis and decreased uric acid clearance.

Chronic elevated serum uric acid (chronic hyperuricemia), typicallydefined as serum urate levels greater than 6.8 mg/dl (greater than 360mmol/), the level above which the physiological saturation threshold isexceeded (Mandell, Cleve. Clin. Med. 75:S5-S8, 2008), is associated witha number of diseases. For example, gout is characterized by recurrentattacks of acute inflammatory arthritis that is caused by aninflammatory reaction to uric acid crystals in the joint typically dueto insufficient renal clearance of uric acid or excessive uric acidproduction. Fructose associated gout is associated with variants oftransporters expressed in the kidney, intestine, and liver. Chronicelevated uric acid is also associated with non-alcoholic steatohepatitis(NASH), non-alcoholic fatty liver disease (NAFLD), metabolic disorder,cardiovascular disease, type 2 diabetes, and conditions linked tooxidative stress, chronic low grade inflammation, and insulin resistance(Xu et al., J. Hepatol. 62:1412-1419, 2015; Cardoso et al., J. Pediatr.89:412-418, 2013; Sertoglu et al., Clin. Biochem., 47:383-388, 2014).

Uric acid (also referred to herein as urate) is the final metabolite ofendogenous and dietary purine metabolism. Xanthine oxidase (XO) (EC1.1.3.22) and xanthine dehydrogenase (XDH) (EC 1.17.1.4), which catalyzethe oxidation of hypoxanthine to xanthine, and xanthine to uric acid,respectively, are interconvertible forms of the same enzyme. The enzymesare molybdopterin-containing flavoproteins that consist of two identicalsubunits of approximately 145 kDa. The enzyme from mammalian sources,including man, is synthesized as the dehydrogenase form, but it can bereadily converted to the oxidase form by oxidation of sulfhydrylresidues or by proteolysis. XDH is primarily expressed in the intestineand the liver, but it is also expressed in other tissues includingadipose tissue.

Allopurinol and febuxostat (Uloric®), inhibitors of the XDH form of theenzyme, are commonly used for the treatment of gout. However, their useis contraindicated in patients with co-morbidities common to gout,especially decreased renal function, e.g., due to chronic kidney diseaseor hepatic impairment. Their use may also be limited in patients withmetabolic syndrome, hypertension, dyslipidemia, non-alcoholicsteatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD),cardiovascular disease, or diabetes (either type 1 or type 2), due tolimited organ function from the disease or condition, or due to adversedrug interactions with agents used for the treatment of such conditions.

Currently, treatments for gout do not fully meet patient needs.Therefore, there is a need for additional therapies for subjects thatwould benefit from reduction in the expression of an XDH gene, such as asubject having an XDH-associated disease or disorder, e.g., gout.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which affect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of a gene encoding xanthine dehydrogenase (XDH). The XDH maybe within a cell, e.g., a cell within a subject, such as a humansubject.

Accordingly, in one aspect the invention provides a double strandedribonucleic acid (dsRNA) agent for inhibiting expression of XDH in acell, wherein the dsRNA agent comprises a sense strand and an antisensestrand forming a double stranded region, wherein the sense strandcomprises at least 15 contiguous nucleotides differing by no more than0, 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO:1and the antisense strand comprises at least 15 contiguous nucleotidesdiffering by no more than 1, 2, or 3 nucleotides from the nucleotidesequence of SEQ ID NO:2. In certain embodiments, the sense strandcomprises at least 15 contiguous nucleotides of the nucleotide sequenceof SEQ ID NO:1 and the antisense strand comprises at least 15 contiguousnucleotides of the nucleotide sequence of SEQ ID NO:2. In certainembodiments, the sense strand comprises at least 17 contiguousnucleotides of the nucleotide sequence of SEQ ID NO:1 and the antisensestrand comprises at least 17 contiguous nucleotides of the nucleotidesequence of SEQ ID NO:2. In certain embodiments, the sense strandcomprises at least 19 contiguous nucleotides of the nucleotide sequenceof SEQ ID NO:1 and the antisense strand comprises at least 19 contiguousnucleotides of the nucleotide sequence of SEQ ID NO:2.

In another aspect, the present invention provides a double strandedribonucleic acid (dsRNA) for inhibiting expression of xanthinedehydrogenase (XDH) in a cell, wherein said dsRNA comprises a sensestrand and an antisense strand forming a double stranded region, whereinthe antisense strand comprises a region of complementarity to an mRNAencoding XDH, and wherein the region of complementarity comprises atleast 15 contiguous nucleotides differing by no more than 0, 1, 2, or 3nucleotides from any one of the antisense nucleotide sequences in anyone of Tables 2-3 and 6-7. In certain embodiments, the region ofcomplementarity comprises at least 15 contiguous nucleotides of any oneof the antisense nucleotide sequences in any one of Tables 2-3 and 6-7.In certain embodiments, the region of complementarity comprises at least17 contiguous nucleotides of any one of the antisense nucleotidesequences in any one of Tables 2-3 and 6-7. In certain embodiments, theregion of complementarity comprises at least 19 contiguous nucleotidesof any one of the antisense nucleotide sequences in any one of Tables2-3 and 6-7. In certain embodiments, the region of complementaritycomprises at least 20 contiguous nucleotides of any one of the antisensenucleotide sequences in any one of Tables 2-3 and 6-7. In certainembodiments, the region of complementarity comprises at least 21contiguous nucleotides of any one of the antisense nucleotide sequencesin any one of Tables 2-3 and 6-7.

In one aspect, the present invention provides a double strandedribonucleic acid (dsRNA) for inhibiting expression of xanthinedehydrogenase (XDH) in a cell, wherein said dsRNA comprises a sensestrand and an antisense strand forming a double stranded region, whereinthe sense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from any one of the nucleotide sequencesof nucleotides 226-269; 1318-1352; 1953-1998; 2351-2394; 2679-2730;3867-3916; or 4510-4574 of SEQ ID NO: 1, and the antisense strandcomprises at least 15 contiguous nucleotides from the correspondingnucleotide sequence of SEQ ID NO: 2.

In one aspect, the present invention provides a double strandedribonucleic acid (dsRNA) for inhibiting expression of xanthinedehydrogenase (XDH) in a cell, wherein said dsRNA comprises a sensestrand and an antisense strand forming a double stranded region, whereinthe sense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from any one of the nucleotide sequencesof nucleotides 15-45; 121-162; 226-292; 306-338; 379-402; 428-458;495-521; 873-907; 1318-1344; 1381-1407; 1604-1643; 1700-1723; 1960-1991;1996-2029; 2044-2067; 2128-2174; 2186-2208; 2289-2345; 2359-2419;2689-2722; 2699-2721; 2774-2797; 2930-2958; 2987-3064; 3083-3158;3195-3221; 3248-3293; 3352-3405; 3460-3520; 3524-3571; 3575-3644;3870-3963; 4143-4176; 4259-4315; 4355-4379; 4395-4467; 4502-4562;4577-4648; 4658-4752; 4815-4854; 5199-5277; 5284-5310; 5358-5446; or5677-5713 of SEQ ID NO: 1, and the antisense strand comprises at least15 contiguous nucleotides from the corresponding nucleotide sequence ofSEQ ID NO: 2.

In one aspect, the present invention provides a double strandedribonucleic acid (dsRNA) for inhibiting expression of xanthinedehydrogenase (XDH) in a cell, wherein said dsRNA comprises a sensestrand and an antisense strand forming a double stranded region, whereinthe sense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from any one of the nucleotide sequencesof nucleotides 123-143; 229-249; 229-251, 230-250; 237-259; 241-263;271-291; 1320-1342; 1321-1341; 1965-1987; 1971-1993; 2130-2150;2682-2704; 2689-2711; 2690-2710; 2699-2721; 3880-3900; or 3883-3903 ofSEQ ID NO: 1, and the antisense strand comprises at least 15 contiguousnucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2.

In some embodiments, the antisense strand comprises at least 15contiguous nucleotides differing by nor more than three nucleotides fromany one of the antisense strand nucleotide sequences of a duplexselected from the group consisting of AD-1395794.1; AD-1136038.3;AD-1395797.1; AD-1135991.3; AD-1297597.2; AD-1395803.1; AD-1395805.1;AD-1395807.1; AD-1395811.1; AD-1297663.2; AD-1136008.2; AD-1395816.1;AD-1136061.2; AD-1135987.2; AD-1395823.1; AD-1136166.3; and AD-1136169.

In some embodiments, the antisense strand comprises at least 15contiguous nucleotides differing by no more than three nucleotides fromany one of the antisense strand nucleotide sequences of a duplexselected from the group consisting of AD-1136091, AD-1395794,AD-1395805, AD-1136008, AD-1136038, AD-1395823, AD-1395816, AD-1136061,AD-1395811, and AD-1136166.

In some embodiments, the sense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequences of nucleotides 2699-2721 of SEQ ID NO:1, and the antisensestrand comprises at least 15 contiguous nucleotides from thecorresponding nucleotide sequence of SEQ ID NO: 2.

In some embodiments, the antisense strand comprises at least 15contiguous nucleotides differing by no more than three nucleotides fromthe antisense strand nucleotide sequence of duplex AD-1136091.

In one embodiment, the dsRNA agent comprises at least one modifiednucleotide.

In one embodiment, substantially all of the nucleotides of the sensestrand; substantially all of the nucleotides of the antisense strandcomprise a modification; or substantially all of the nucleotides of thesense strand and substantially all of the nucleotides of the antisensestrand comprise a modification.

In one embodiment, all of the nucleotides of the sense strand comprise amodification; all of the nucleotides of the antisense strand comprise amodification; or all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand comprise a modification.

In one embodiment, at least one of the modified nucleotides is selectedfrom the group consisting of a deoxy-nucleotide, a 3′-terminaldeoxythimidine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a lockednucleotide, an unlocked nucleotide, a conformationally restrictednucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide,2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga phosphorothioate group, a nucleotide comprising a methylphosphonategroup, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a5′-phosphate mimic, a nucleotide comprising a 2′-phosphate, e.g.,cytidine-2′-phosphate (C2p); guanosine-2′-phosphate (G2p);uridine-2′-phosphate (U2p); adenosine-2′-phosphate (A2p); a thermallydestabilizing nucleotide, a glycol modified nucleotide (GNA), and a2-O—(N-methylacetamide) modified nucleotide; and combinations thereof.

In one embodiment, the modifications on the nucleotides are selectedfrom the group consisting of LNA, glycol nucleic acid (GNA), hexitolnucleic acid (HNA), CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C—allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and glycol; and combinationsthereof.

In one embodiment, at least one of the modified nucleotides is selectedfrom the group consisting of a deoxy-nucleotide, a 2′-O-methyl modifiednucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a nucleotide comprising a 2′-phosphate, a glycol modifiednucleotide (GNA), e.g., Ggn, Cgn, Tgn, or Agn, and, a vinyl-phosphonatenucleotide; and combinations thereof.

In another embodiment, at least one of the modifications on thenucleotides is a thermally destabilizing nucleotide modification.

In one embodiment, the thermally destabilizing nucleotide modificationis selected from the group consisting of an abasic modification; amismatch with the opposing nucleotide in the duplex; and destabilizingsugar modification, a 2′-deoxy modification, an acyclic nucleotide, anunlocked nucleic acid (UNA), and a glycerol nucleic acid (GNA).

The double stranded region may be 19-30 nucleotide pairs in length;19-25 nucleotide pairs in length; 19-23 nucleotide pairs in length;23-27 nucleotide pairs in length; or 21-23 nucleotide pairs in length.

In one embodiment, each strand is independently no more than 30nucleotides in length.

In one embodiment, the sense strand is 21 nucleotides in length and theantisense strand is 23 nucleotides in length.

The region of complementarity may be at least 17 nucleotides in length;19-23 nucleotides in length; or 19 nucleotides in length.

In one embodiment, at least one strand comprises a 3′ overhang of atleast 1 nucleotide. In another embodiment, at least one strand comprisesa 3′ overhang of at least 2 nucleotides.

In one embodiment, the dsRNA agent further comprises a ligand.

In one embodiment, the ligand is conjugated to the 3′ end of the sensestrand of the dsRNA agent.

In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc)derivative.

In one embodiment, the ligand is one or more GalNAc derivatives attachedthrough a monovalent, bivalent, or trivalent branched linker.

In one embodiment, the ligand is

In one embodiment, the dsRNA agent is conjugated to the ligand as shownin the following schematic

and, wherein X is O or S.

In one embodiment, the X is O.

In one embodiment, the dsRNA agent further comprises at least onephosphorothioate or methylphosphonate internucleotide linkage.

In one embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 3′-terminus of one strand, e.g., theantisense strand or the sense strand.

In another embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 5′-terminus of one strand, e.g., theantisense strand or the sense strand.

In one embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the both the 5′- and 3′-terminus of onestrand. In one embodiment, the strand is the antisense strand.

In one embodiment, the base pair at the 1 position of the 5′-end of theantisense strand of the duplex is an AU base pair.

The present invention also provides cells containing any of the dsRNAagents of the invention and pharmaceutical compositions comprising anyof the dsRNA agents of the invention.

The pharmaceutical composition of the invention may include dsRNA agentin an unbuffered solution, e.g., saline or water, or the pharmaceuticalcomposition of the invention may include the dsRNA agent is in a buffersolution, e.g., a buffer solution comprising acetate, citrate,prolamine, carbonate, or phosphate or any combination thereof; orphosphate buffered saline (PBS).

In one aspect, the present invention provides a method of inhibitingexpression of a xanthine dehydrogenase (XDH) gene in a cell. The methodincludes contacting the cell with any of the dsRNAs of the invention orany of the pharmaceutical compositions of the invention, therebyinhibiting expression of the XDH gene in the cell.

In one embodiment, the cell is within a subject, e.g., a human subject,e.g., a subject having a xanthine dehydrogenase-(XDH)-associateddisease. Such diseases are typically associated with excess uric acid,e.g., serum uric acid.

In one embodiment, the XDH-associated disease is hyperuricemia. Inanother embodiment, the XDH-associated disease is gout.

In one embodiment, contacting the cell with the dsRNA agent inhibits theexpression of XDH by at least 50%, 60%, 70%, 80%, 90%, or 95%.

In one embodiment, inhibiting expression of XDH decreases XDH proteinlevel in serum of the subject by at least 50%, 60%, 70%, 80%, 90%, or95%.

In one aspect, the present invention provides a method of treating asubject having a disorder that would benefit from reduction in xanthinedehydrogenase (XDH) expression. The method includes administering to thesubject a therapeutically effective amount of any of the dsRNAs of theinvention or any of the pharmaceutical compositions of the invention,thereby treating the subject having the disorder that would benefit fromreduction in XDH expression.

In another aspect, the present invention provides a method of preventingdevelopment of a disorder that would benefit from reduction in xanthinedehydrogenase (XDH) expression in a subject having at least one sign orsymptom of a disorder who does not yet meet the diagnostic criteria forthat disorder. The method includes administering to the subject aprophylactically effective amount of any of the dsRNAs of the inventionor any of the pharmaceutical compositions of the invention, therebypreventing the subject progressing to meet the diagnostic criteria ofthe disorder that would benefit from reduction in XDH expression.

In one embodiment, the disorder is a xanthinedehydrogenase-(XDH)-associated disorder.

In one embodiment, the subject is human.

In one embodiment, the dsRNA agent is administered to the subject at adose of about 0.01 mg/kg to about 50 mg/kg.

In one embodiment, the dsRNA agent is administered to the subjectsubcutaneously.

In one embodiment, the level of XDH in the subject sample(s) is an XDHprotein level in a blood or serum sample(s).

In one embodiment, the administration of the agent to the subject causesa decrease in the level of uric acid (e.g., serum uric acid) or adecrease in XDH protein accumulation.

In certain embodiments, the methods of the invention further compriseadministering to the subject an additional therapeutic agent.

In certain embodiments, the compositions and methods of the inventionare used in combination with other compositions and methods to treathyperuricemia and/or gout, e.g., allopurinol, oxypurinol, febuxostat,analgesic or anti-inflammatory agents, e.g., NSAIDS.

The present invention also provides kits comprising any of the dsRNAs ofthe invention or any of the pharmaceutical compositions of theinvention, and optionally, instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the uric acid metabolic pathway. XDH islabeled as XO in the schematic.

FIG. 2 depicts the levels of XDH protein in liver samples obtained fromCynomolgus monkeys following treatment with the indicated XDH targetingsiRNAs or allopurinol.

FIG. 3 depicts the levels of levels of XDH mRNA in liver samplesobtained from Cynomolgus monkeys following treatment with the indicatedXDH targeting siRNAs or allopurinol.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of a xanthine dehydrogenase (XDH) gene.

The gene may be within a cell, e.g., a cell within a subject, such as ahuman. The use of these iRNAs enables the targeted degradation of mRNAsof the corresponding gene (xanthine dehydrogenase gene) in mammals.

The iRNAs of the invention have been designed to target the humanxanthine dehydrogenase gene, including portions of the gene that areconserved in the xanthine dehydrogenase orthologs of other mammalianspecies. Without intending to be limited by theory, it is believed thata combination or sub-combination of the foregoing properties and thespecific target sites or the specific modifications in these iRNAsconfer to the iRNAs of the invention improved efficacy, stability,potency, durability, and safety.

Accordingly, the present invention provides methods for treating andpreventing a xanthine dehydrogenase-associated disorder, disease, orcondition, e.g., a disorder, disease, or condition associated withelevated serum uric acid levels, e.g., hyperuricemia and gout, usingiRNA compositions which effect the RNA-induced silencing complex(RISC)-mediated cleavage of RNA transcripts of a xanthine dehydrogenasegene.

The iRNAs of the invention include an RNA strand (the antisense strand)having a region which is up to about 30 nucleotides or less in length,e.g., 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22,19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23,20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or21-22 nucleotides in length, which region is substantially complementaryto at least part of an mRNA transcript of a xanthine dehydrogenase gene.In certain embodiments, the RNAi agents of the disclosure include an RNAstrand (the antisense strand) having a region which is about 21-23nucleotides in length, which region is substantially complementary to atleast part of an mRNA transcript of a xanthine dehydrogenase gene.

In certain embodiments, one or both of the strands of the doublestranded RNAi agents of the invention is up to 66 nucleotides in length,e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length,with a region of at least 19 contiguous nucleotides that issubstantially complementary to at least a part of an mRNA transcript ofa xanthine dehydrogenase gene. In some embodiments, such iRNA agentshaving longer length antisense strands can, for example, include asecond RNA strand (the sense strand) of 20-60 nucleotides in lengthwherein the sense and antisense strands form a duplex of 18-30contiguous nucleotides.

The use of iRNAs of the invention enables the targeted degradation ofmRNAs of the corresponding gene (xanthine dehydrogenase gene) inmammals. Using in vitro and in vivo assays, the present inventors havedemonstrated that iRNAs targeting a xanthine dehydrogenase gene canpotently mediate RNAi, resulting in significant inhibition of expressionof a xanthine dehydrogenase gene. Thus, methods and compositionsincluding these iRNAs are useful for treating a subject having axanthine dehydrogenase-associated disorder, e.g., hyperuricemia andgout.

Accordingly, the present invention provides methods and combinationtherapies for treating a subject having a disorder that would benefitfrom inhibiting or reducing the expression of a xanthine dehydrogenasegene, e.g., a xanthine dehydrogenase-associated disease, such ashyperuricemia and gout, using iRNA compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of an XDH gene.

The present invention also provides methods for preventing at least onesymptom in a subject having a disorder that would benefit frominhibiting or reducing the expression of a xanthine dehydrogenase gene,e.g., hyperuricemia and gout.

In certain embodiments, the administration of the dsRNA to the subjectcauses a decrease in XDH mRNA level, XDH protein level, and the level ofserum uric acid.

The following detailed description discloses how to make and usecompositions containing iRNAs to inhibit the expression of a xanthinedehydrogenase gene as well as compositions, uses, and methods fortreating subjects that would benefit from inhibition or reduction of theexpression of a xanthine dehydrogenase gene, e.g., subjects susceptibleto or diagnosed with a xanthine dehydrogenase-associated disorder.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise. Forexample, “sense strand or antisense strand” is understood as “sensestrand or antisense strand or sense strand and antisense strand.”

The term “about” is used herein to mean within the typical ranges oftolerances in the art. For example, “about” can be understood as about 2standard deviations from the mean. In certain embodiments, aboutmeans±10%. In certain embodiments, about means±5%. When about is presentbefore a series of numbers or a range, it is understood that “about” canmodify each of the numbers in the series or range.

The term “at least”, “no less than”, or “or more” prior to a number orseries of numbers is understood to include the number adjacent to theterm “at least”, and all subsequent numbers or integers that couldlogically be included, as clear from context. For example, the number ofnucleotides in a nucleic acid molecule must be an integer. For example,“at least 19 nucleotides of a 21 nucleotide nucleic acid molecule” meansthat 19, 20, or 21 nucleotides have the indicated property. When atleast is present before a series of numbers or a range, it is understoodthat “at least” can modify each of the numbers in the series or range.

As used herein, “no more than” or “or less” is understood as the valueadjacent to the phrase and logical lower values or integers, as logicalfrom context, to zero. For example, a duplex with an overhang of “nomore than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “nomore than” is present before a series of numbers or a range, it isunderstood that “no more than” can modify each of the numbers in theseries or range. As used herein, ranges include both the upper and lowerlimit.

As used herein, methods of detection can include determination that theamount of analyte present is below the level of detection of the method.

In the event of a conflict between an indicated target site and thenucleotide sequence for a sense or antisense strand, the indicatedsequence takes precedence.

In the event of a conflict between a sequence and its indicated site ona transcript or other sequence, the nucleotide sequence recited in thespecification takes precedence.

As used herein, the term “xanthine dehydrogenase,” used interchangeablywith the term “XDH,” refers to the well-known gene and polypeptide, alsoknown in the art as Xanthine Dehydrogenase/Oxidase, XanthineOxidoreductase, XAN1, XDHA, XOR, XO, EC 1.17.1.4, and EC 1.7.2.2.

XDH belongs to the group of molybdenum-containing hydroxylases involvedin the oxidative metabolism of purines. The encoded protein has beenidentified as a moonlighting protein based on its ability to performmechanistically distinct functions. Xanthine dehydrogenase can beconverted to xanthine oxidase by reversible sulfhydryl oxidation or byirreversible proteolytic modification. As used herein, unless clear fromcontext, xanthine dehydrogenase or XDH is understood to include both thexanthine dehydrogenase and xanthine oxidase (“XO” or “XOR”) form of theprotein. The protein is expressed predominantly in the intestine and theliver, but is also expressed in adipose tissue. Two transcript variantshave been identified for the human isoform of the gene.

The term “XDH” includes human XDH, the amino acid and nucleotidesequence of which may be found in, for example, GenBank Accession Nos.GI:91823270, GI: 1519244313, and GI:767915203; mouse XDH, the amino acidand nucleotide sequence of which may be found in, for example, GenBankAccession No. GI:575501724; rat XDH, the amino acid and nucleotidesequence of which may be found in, for example, GenBank Accession No.GI:8394543; and Macaca fascicularis XDH, transcript variant X1, theamino acid and nucleotide sequence of which may be found in, forexample, GenBank Accession Nos. GI:544482046 and GI:544482048.Additional examples of XDH mRNA sequences are readily available using,e.g., GenBank, UniProt, OMIM, and the Macaca genome project web site.

Exemplary XDH nucleotide sequences may also be found in SEQ ID NOs:1, 3,5, 7, 9, 11, and 15. SEQ ID NOs:2, 4, 6, 8, 10, 12, and 16 are theantisense sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 15,respectively.

The term “XDH,” as used herein, also refers to naturally occurring DNAsequence variations of the XDH gene. The term “XDH,” as used herein,also refers to single nucleotide polymorphisms in the XDH gene. Numeroussequence variations within the XDH gene have been identified and may befound at, for example, NCBI dbSNP and UniProt (see, e.g.,www.ncbi.nlm.nih.gov/snp?LinkName=gene_snp&from_uid=7498 (which isincorporated herein by reference as of the date of filing thisapplication) which lists over 3000 SNPs in human XDH). In certainembodiments, such naturally occurring variants are included within thescope of the XDH gene sequence.

Further information on XDH can be found, for example, atwww.ncbi.nlm.nih.gov/gene/7498 (which is incorporated herein byreference as of the date of filing this application).

Additional examples of XDH mRNA sequences are readily available throughpublicly available databases, e.g., GenBank, UniProt, OMIM, and theMacaca genome project web site.

The entire contents of each of the foregoing GenBank Accession numbersand the Gene database numbers are incorporated herein by reference as ofthe date of filing this application.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a xanthine dehydrogenase gene, including mRNA that is a product ofRNA processing of a primary transcription product. The target portion ofthe sequence will be at least long enough to serve as a substrate foriRNA-directed cleavage at or near that portion of the nucleotidesequence of an mRNA molecule formed during the transcription of an XDHgene. In one embodiment, the target sequence is within the proteincoding region of XDH.

The target sequence may be from about 19-36 nucleotides in length, e.g.,about 19-30 nucleotides in length. For example, the target sequence canbe about 19-30 nucleotides, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25,19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26,20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26,21-25, 21-24, 21-23, or 21-22 nucleotides in length. In someembodiments, the target sequence is about 19 to about 30 nucleotides inlength. In other embodiments, the target sequence is about 19 to about25 nucleotides in length. In still other embodiments, the targetsequence is about 19 to about 23 nucleotides in length. In someembodiments, the target sequence is about 21 to about 23 nucleotides inlength. Ranges and lengths intermediate to the above recited ranges andlengths are also contemplated to be part of the invention.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine, and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacement moiety(see, e.g., Table 1). The skilled person is well aware that guanine,cytosine, adenine, and uracil can be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base can basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent”as used interchangeably herein, refer to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.iRNA directs the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The iRNA modulates, e.g., inhibits,the expression of a xanthine dehydrogenase gene in a cell, e.g., a cellwithin a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a singlestranded RNA that interacts with a target RNA sequence, e.g., a xanthinedehydrogenase target mRNA sequence, to direct the cleavage of the targetRNA. Without wishing to be bound by theory it is believed that longdouble stranded RNA introduced into cells is broken down into siRNA by aType III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev.15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into19-23 base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect theinvention relates to a single stranded RNA (siRNA) generated within acell and which promotes the formation of a RISC complex to effectsilencing of the target gene, i.e., a xanthine dehydrogenase (XDH) gene.Accordingly, the term “siRNA” is also used herein to refer to an iRNA asdescribed above.

In certain embodiments, the RNAi agent may be a single-stranded siRNA(ssRNAi) that is introduced into a cell or organism to inhibit a targetmRNA. Single-stranded RNAi agents bind to the RISC endonuclease,Argonaute 2, which then cleaves the target mRNA. The single-strandedsiRNAs are generally 15-30 nucleotides and are chemically modified. Thedesign and testing of single-stranded siRNAs are described in U.S. Pat.No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entirecontents of each of which are hereby incorporated herein by reference.Any of the antisense nucleotide sequences described herein may be usedas a single-stranded siRNA as described herein or as chemically modifiedby the methods described in Lima et al., (2012) Cell 150:883-894.

In certain embodiments, an “iRNA” for use in the compositions, uses, andmethods of the invention is a double stranded RNA and is referred toherein as a “double stranded RNA agent,” “double stranded RNA (dsRNA)molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary nucleicacid strands, referred to as having “sense” and “antisense” orientationswith respect to a target RNA, i.e., a xanthine dehydrogenase (XDH) gene.In some embodiments of the invention, a double stranded RNA (dsRNA)triggers the degradation of a target RNA, e.g., an mRNA, through apost-transcriptional gene-silencing mechanism referred to herein as RNAinterference or RNAi.

As used herein, the term “modified nucleotide” refers to a nucleotidehaving, independently, a modified sugar moiety, a modifiedinternucleotide linkage, or modified nucleobase, or any combinationthereof. Thus, the term modified nucleotide encompasses substitutions,additions or removal of, e.g., a functional group or atom, tointernucleoside linkages, sugar moieties, or nucleobases. Themodifications suitable for use in the agents of the invention includeall types of modifications disclosed herein or known in the art. Anysuch modifications, as used in a siRNA type molecule, are encompassed by“iRNA” or “RNAi agent” for the purposes of this specification andclaims.

In certain embodiments of the instant disclosure, inclusion of adeoxy-nucleotide—which is acknowledged as a naturally occurring form ofnucleotide—if present within a RNAi agent can be considered toconstitute a modified nucleotide.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about19 to 36 base pairs in length, e.g., about 19-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairsin length, such as about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25,19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26,20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26,21-25, 21-24, 21-23, or 21-22 base pairs in length. In certainembodiments, the duplex region is 19-21 base pairs in length, e.g., 21base pairs in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of thedisclosure.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome embodiments, the hairpin loop can comprise at least 4, 5, 6, 7, 8,9, 10, 20, 23 or more unpaired nucleotides. In some embodiments, thehairpin loop can be 10 or fewer nucleotides.

In some embodiments, the hairpin loop can be 8 or fewer unpairednucleotides. In some embodiments, the hairpin loop can be 4-10 unpairednucleotides. In some embodiments, the hairpin loop can be 4-8nucleotides.

In certain embodiment, the two strands of double-stranded oligomericcompound can be linked together. The two strands can be linked to eachother at both ends, or at one end only. By linking at one end is meantthat 5′-end of first strand is linked to the 3′-end of the second strandor 3′-end of first strand is linked to 5′-end of the second strand. Whenthe two strands are linked to each other at both ends, 5′-end of firststrand is linked to 3′-end of second strand and 3′-end of first strandis linked to 5′-end of second strand. The two strands can be linkedtogether by an oligonucleotide linker including, but not limited to,(N)n; wherein N is independently a modified or unmodified nucleotide andn is 3-23. In some embodiments, n is 3-10, e.g., 3, 4, 5, 6, 7, 8, 9, or10. In some embodiments, the oligonucleotide linker is selected from thegroup consisting of GNRA, (G)4, (U)4, and (dT)4, wherein N is a modifiedor unmodified nucleotide and R is a modified or unmodified purinenucleotide. Some of the nucleotides in the linker can be involved inbase-pair interactions with other nucleotides in the linker. The twostrands can also be linked together by a non-nucleosidic linker, e.g. alinker described herein. It will be appreciated by one of skill in theart that any oligonucleotide chemical modifications or variationsdescribe herein can be used in the oligonucleotide linker.

Hairpin and dumbbell type oligomeric compounds will have a duplex regionequal to or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or25 nucleotide pairs. The duplex region can be equal to or less than 200,100, or 50, in length. In some embodiments, ranges for the duplex regionare 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.

The hairpin oligomeric compounds can have a single strand overhang orterminal unpaired region, in some embodiments at the 3′, and in someembodiments on the antisense side of the hairpin. In some embodiments,the overhangs are 1-4, more generally 2-3 nucleotides in length. Thehairpin oligomeric compounds that can induce RNA interference are alsoreferred to as “shRNA” herein.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not be, butcan be covalently connected. Where the two strands are connectedcovalently by means other than an uninterrupted chain of nucleotidesbetween the 3′-end of one strand and the 5′-end of the respective otherstrand forming the duplex structure, the connecting structure isreferred to as a “linker.” The RNA strands may have the same or adifferent number of nucleotides. The maximum number of base pairs is thenumber of nucleotides in the shortest strand of the dsRNA minus anyoverhangs that are present in the duplex. In addition to the duplexstructure, an RNAi may comprise one or more nucleotide overhangs. In oneembodiment of the RNAi agent, at least one strand comprises a 3′overhang of at least 1 nucleotide. In another embodiment, at least onestrand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4,5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments,at least one strand of the RNAi agent comprises a 5′ overhang of atleast 1 nucleotide. In certain embodiments, at least one strandcomprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6,7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments,both the 3′ and the 5′ end of one strand of the RNAi agent comprise anoverhang of at least 1 nucleotide.

In certain embodiments, an iRNA agent of the invention is a dsRNA, eachstrand of which comprises 19-23 nucleotides, that interacts with atarget RNA sequence, e.g., a xanthine dehydrogenase (XDH) gene, todirect cleavage of the target RNA.

In some embodiments, an iRNA of the invention is a dsRNA of 24-30nucleotides that interacts with a target RNA sequence, e.g., an XDHtarget mRNA sequence, to direct the cleavage of the target RNA.

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of a doublestranded iRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively, the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand, or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′-end, 3′-end, orboth ends of either an antisense or sense strand of a dsRNA.

In one embodiment of the dsRNA, at least one strand comprises a 3′overhang of at least 1 nucleotide. In another embodiment, at least onestrand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4,5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments,at least one strand of the RNAi agent comprises a 5′ overhang of atleast 1 nucleotide. In certain embodiments, at least one strandcomprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6,7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments,both the 3′ and the 5′ end of one strand of the RNAi agent comprise anoverhang of at least 1 nucleotide.

In one embodiment, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide,overhang at the 3′-end or the 5′-end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In anotherembodiment, one or more of the nucleotides in the overhang is replacedwith a nucleoside thiophosphate.

In certain embodiments, the antisense strand of a dsRNA has a 1-10nucleotides, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide,overhang at the 3′-end or the 5′-end. In certain embodiments, theoverhang on the sense strand or the antisense strand, or both, caninclude extended lengths longer than 10 nucleotides, e.g., 1-30nucleotides, 2-30 nucleotides, 10-30 nucleotides, 10-25 nucleotides,10-20 nucleotides, or 10-15 nucleotides in length. In certainembodiments, an extended overhang is on the sense strand of the duplex.In certain embodiments, an extended overhang is present on the 3′ end ofthe sense strand of the duplex. In certain embodiments, an extendedoverhang is present on the 5′ end of the sense strand of the duplex. Incertain embodiments, an extended overhang is on the antisense strand ofthe duplex. In certain embodiments, an extended overhang is present onthe 3′end of the antisense strand of the duplex. In certain embodiments,an extended overhang is present on the 5′end of the antisense strand ofthe duplex. In certain embodiments, one or more of the nucleotides inthe extended overhang is replaced with a nucleoside thiophosphate. Incertain embodiments, the overhang includes a self-complementary portionsuch that the overhang is capable of forming a hairpin structure that isstable under physiological conditions.

“Blunt” or “blunt end” means that there are no unpaired nucleotides atthat end of the double stranded RNA agent, i.e., no nucleotide overhang.A “blunt ended” double stranded RNA agent is double stranded over itsentire length, i.e., no nucleotide overhang at either end of themolecule. The RNAi agents of the invention include RNAi agents with nonucleotide overhang at one end (i.e., agents with one overhang and oneblunt end) or with no nucleotide overhangs at either end. Most oftensuch a molecule will be double-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., an XDH mRNA.

As used herein, the term “region of complementarity” refers to theregion on the antisense strand that is substantially complementary to asequence, for example a target sequence, e.g., a xanthine dehydrogenasenucleotide sequence, as defined herein. Where the region ofcomplementarity is not fully complementary to the target sequence, themismatches can be in the internal or terminal regions of the molecule.Generally, the most tolerated mismatches are in the terminal regions,e.g., within 5, 4, or 3 nucleotides of the 5′- or 3′-end of the iRNA. Insome embodiments, a double stranded RNA agent of the invention includesa nucleotide mismatch in the antisense strand. In some embodiments, theantisense strand of the double stranded RNA agent of the inventionincludes no more than 4 mismatches with the target mRNA, e.g., theantisense strand includes 4, 3, 2, 1, or 0 mismatches with the targetmRNA. In some embodiments, the antisense strand double stranded RNAagent of the invention includes no more than 4 mismatches with the sensestrand, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatcheswith the sense strand. In some embodiments, a double stranded RNA agentof the invention includes a nucleotide mismatch in the sense strand. Insome embodiments, the sense strand of the double stranded RNA agent ofthe invention includes no more than 4 mismatches with the antisensestrand, e.g., the sense strand includes 4, 3, 2, 1, or 0 mismatches withthe antisense strand. In some embodiments, the nucleotide mismatch is,for example, within 5, 4, 3 nucleotides from the 3′-end of the iRNA. Inanother embodiment, the nucleotide mismatch is, for example, in the3′-terminal nucleotide of the iRNA agent. In some embodiments, themismatch(s) is not in the seed region.

Thus, an RNAi agent as described herein can contain one or moremismatches to the target sequence. In one embodiment, a RNAi agent asdescribed herein contains no more than 3 mismatches (i.e., 3, 2, 1, or 0mismatches). In one embodiment, an RNAi agent as described hereincontains no more than 2 mismatches. In one embodiment, an RNAi agent asdescribed herein contains no more than 1 mismatch. In one embodiment, anRNAi agent as described herein contains 0 mismatches. In certainembodiments, if the antisense strand of the RNAi agent containsmismatches to the target sequence, the mismatch can optionally berestricted to be within the last 5 nucleotides from either the 5′- or3′-end of the region of complementarity. For example, in suchembodiments, for a 23 nucleotide RNAi agent, the strand which iscomplementary to a region of an XDH gene, generally does not contain anymismatch within the central 13 nucleotides. The methods described hereinor methods known in the art can be used to determine whether an RNAiagent containing a mismatch to a target sequence is effective ininhibiting the expression of an XDH gene. Consideration of the efficacyof RNAi agents with mismatches in inhibiting expression of an XDH geneis important, especially if the particular region of complementarity inan XDH gene is known to have polymorphic sequence variation within thepopulation.

The term “sense strand” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, “substantially all of the nucleotides are modified” arelargely but not wholly modified and can include not more than 5, 4, 3,2, or 1 unmodified nucleotides.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can be, for example, “stringent conditions”, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g.,“Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) ColdSpring Harbor Laboratory Press). Other conditions, such asphysiologically relevant conditions as can be encountered inside anorganism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3, or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression, in vitro orin vivo. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs or base pairs formedfrom non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogsteen base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweentwo oligonucleotides or polynucleotides, such as the antisense strand ofa double stranded RNA agent and a target sequence, as will be understoodfrom the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding a xanthine dehydrogenase gene). Forexample, a polynucleotide is complementary to at least a part of axanthine dehydrogenase mRNA if the sequence is substantiallycomplementary to a non-interrupted portion of an mRNA encoding axanthine dehydrogenase gene.

Accordingly, in some embodiments, the antisense polynucleotidesdisclosed herein are fully complementary to the target XDH sequence.

In other embodiments, the antisense polynucleotides disclosed herein aresubstantially complementary to the target XDH sequence and comprise acontiguous nucleotide sequence which is at least 80% complementary overits entire length to the equivalent region of the nucleotide sequence ofany one of SEQ ID NOs:1, 3, 5, 7, 9, 11, or 15, or a fragment of any oneof SEQ ID NOs:1, 3, 5, 7, 9, 11, or 15, such as about 85%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, or about 99% complementary.

In other embodiments, the antisense polynucleotides disclosed herein aresubstantially complementary to the target XDH sequence and comprise acontiguous nucleotide sequence which is at least about 80% complementaryover its entire length to any one of the sense strand nucleotidesequences in any one of Tables 2-3 and 6-7, or a fragment of any one ofthe sense strand nucleotide sequences in any one of Tables 2-3 and 6-7,such as about 85%, about 90%, about 95%, or fully complementary.

In some embodiments, the antisense polynucleotides disclosed herein aresubstantially complementary to the target XDH sequence and comprise acontiguous nucleotide sequence which is at least about 80% complementaryover its entire length to a fragment of SEQ ID NO:1 selected from thegroup of nucleotides 226-269; 1318-1352; 1953-1998; 2351-2394;2679-2730; 3867-3916; or 4510-4574 of SEQ ID NO: 1, such as about 85%,about 90%, about 95%, or fully complementary.

In some embodiments, the antisense polynucleotides disclosed herein aresubstantially complementary to the target XDH sequence and comprise acontiguous nucleotide sequence which is at least about 80% complementaryover its entire length to a fragment of SEQ ID NO:1 selected from thegroup of nucleotides 15-45; 121-162; 226-292; 306-338; 379-402; 428-458;495-521; 873-907; 1318-1344; 1381-1407; 1604-1643; 1700-1723; 1960-1991;1996-2029; 2044-2067; 2128-2174; 2186-2208; 2289-2345; 2359-2419;2689-2722; 2699-2721; 2774-2797; 2930-2958; 2987-3064; 3083-3158;3195-3221; 3248-3293; 3352-3405; 3460-3520; 3524-3571; 3575-3644;3870-3963; 4143-4176; 4259-4315; 4355-4379; 4395-4467; 4502-4562;4577-4648; 4658-4752; 4815-4854; 5199-5277; 5284-5310; 5358-5446; or5677-5713 of SEQ ID NO: 1, such as about 85%, about 90%, about 95%, orfully complementary.

In some embodiments, the antisense polynucleotides disclosed herein aresubstantially complementary to the target XDH sequence and comprise acontiguous nucleotide sequence which is at least about 80% complementaryover its entire length to a fragment of SEQ ID NO:1 selected from thegroup of nucleotides 123-143; 229-249; 229-251, 230-250; 237-259;241-263; 271-291; 1320-1342; 1321-1341; 1965-1987; 1971-1993; 2130-2150;2682-2704; 2689-2711; 2690-2710; 2699-2721; 3880-3900; or 3883-3903 ofSEQ ID NO: 1, such as about 85%, about 90%, about 95%, or fullycomplementary.

In some embodiments, the antisense polynucleotides disclosed herein aresubstantially complementary to the target XDH sequence and comprise acontiguous nucleotide sequence which is at least about 80% complementaryover its entire length to a fragment of SEQ ID NO:1, e.g., nucleotides2699-2721 of SEQ ID NO:1, such as about 85%, about 90%, about 95%, orfully complementary.

In one embodiment, an RNAi agent of the disclosure includes a sensestrand that is substantially complementary to an antisensepolynucleotide which, in turn, is the same as a target XDH sequence, andwherein the sense strand polynucleotide comprises a contiguousnucleotide sequence which is at least about 80% complementary over itsentire length to the equivalent region of the nucleotide sequence of SEQID NOs: 2, 4, 6, 8, 10, 12, or 16, or a fragment of any one of SEQ IDNOs:2, 4, 6, 8, 10, 12, or 16, such as about 85%, about 90%, about 95%,or fully complementary.

In some embodiments, an RNAi agent of the invention includes a sensestrand that is substantially complementary to an antisensepolynucleotide which, in turn, is complementary to a target xanthinedehydrogenase sequence, and wherein the sense strand polynucleotidecomprises a contiguous nucleotide sequence which is at least about 80%complementary over its entire length to any one of the antisense strandnucleotide sequences in any one of Tables 2-3 and 6-7, or a fragment ofany one of the antisense strand nucleotide sequences in any one ofTables 2-3 and 6-7, such as about 85%, about 90%, about 95%, or fullycomplementary.

In some embodiments, the double-stranded region of a double-strandediRNA agent is equal to or at least, 17, 18, 19, 20, 21, 22, 23, 23, 24,25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.

In some embodiments, the antisense strand of a double-stranded iRNAagent is equal to or at least 17, 18, 19, 20, 21, 22, 23, 23, 24, 25,26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, the sense strand of a double-stranded iRNA agent isequal to or at least 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28,29, or 30 nucleotides in length.

In one embodiment, the sense and antisense strands of thedouble-stranded iRNA agent are each 18 to 30 nucleotides in length.

In one embodiment, the sense and antisense strands of thedouble-stranded iRNA agent are each 19 to 25 nucleotides in length.

In one embodiment, the sense and antisense strands of thedouble-stranded iRNA agent are each 21 to 23 nucleotides in length.

In one embodiment, the sense strand of the iRNA agent is 21-nucleotidesin length, and the antisense strand is 23-nucleotides in length, whereinthe strands form a double-stranded region of 21 consecutive base pairshaving a 2-nucleotide long single stranded overhangs at the 3′-end.

In some embodiments, the majority of nucleotides of each strand areribonucleotides, but as described in detail herein, each or both strandscan also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide or a modified nucleotide. In addition, an “iRNA” mayinclude ribonucleotides with chemical modifications. Such modificationsmay include all types of modifications disclosed herein or known in theart. Any such modifications, as used in an iRNA molecule, areencompassed by “iRNA” for the purposes of this specification and claims.

In certain embodiments of the instant disclosure, inclusion of adeoxy-nucleotide if present within an RNAi agent can be considered toconstitute a modified nucleotide.

In one embodiment, at least partial suppression of the expression of anXDH gene, is assessed by a reduction of the amount of XDH mRNA which canbe isolated from or detected in a first cell or group of cells in whichan XDH gene is transcribed and which has or have been treated such thatthe expression of an XDH gene is inhibited, as compared to a second cellor group of cells substantially identical to the first cell or group ofcells but which has or have not been so treated (control cells). Thedegree of inhibition may be expressed in terms of:

${\frac{\left( {{mRNA}{in}{control}{cells}} \right) - \left( {{mRNA}{in}{treated}{cells}} \right)}{\left( {{mRNA}{in}{control}{cells}} \right)} \cdot 100}\%$

The phrase “contacting a cell with an iRNA,” such as a dsRNA, as usedherein, includes contacting a cell by any possible means. Contacting acell with an iRNA includes contacting a cell in vitro with the iRNA orcontacting a cell in vivo with the iRNA. The contacting may be donedirectly or indirectly. Thus, for example, the iRNA may be put intophysical contact with the cell by the individual performing the method,or alternatively, the iRNA may be put into a situation that will permitor cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the iRNA. Contacting a cell in vivo may be done, for example,by injecting the iRNA into or near the tissue where the cell is located,or by injecting the iRNA into another area, e.g., the bloodstream or thesubcutaneous space, such that the agent will subsequently reach thetissue where the cell to be contacted is located. For example, the iRNAmay contain or be coupled to a ligand, e.g., GalNAc, that directs theiRNA to a site of interest, e.g., the liver. Combinations of in vitroand in vivo methods of contacting are also possible. For example, a cellmay also be contacted in vitro with an iRNA and subsequentlytransplanted into a subject.

In certain embodiments, contacting a cell with an iRNA includes“introducing” or “delivering the iRNA into the cell” by facilitating oreffecting uptake or absorption into the cell. Absorption or uptake of aniRNA can occur through unaided diffusion or active cellular processes,or by auxiliary agents or devices. Introducing an iRNA into a cell maybe in vitro or in vivo. For example, for in vivo introduction, iRNA canbe injected into a tissue site or administered systemically. In vitrointroduction into a cell includes methods known in the art such aselectroporation and lipofection. Further approaches are described hereinbelow or are known in the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipidlayer encapsulating a pharmaceutically active molecule, such as anucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA istranscribed. LNPs are described in, for example, U.S. Pat. Nos.6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents ofwhich are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a rabbit, a sheep, a hamster, aguinea pig, a dog, a rat, or a mouse), or a bird that expresses thetarget gene, either endogenously or heterologously. In an embodiment,the subject is a human, such as a human being treated or assessed for adisease or disorder that would benefit from reduction in XDH expression;a human at risk for a disease or disorder that would benefit fromreduction in XDH expression; a human having a disease or disorder thatwould benefit from reduction in XDH expression; or human being treatedfor a disease or disorder that would benefit from reduction in XDHexpression as described herein. In some embodiments, the subject is afemale human. In other embodiments, the subject is a male human. In oneembodiment, the subject is an adult subject. In another embodiment, thesubject is a pediatric subject.

As used herein, the terms “treating” or “treatment” refer to abeneficial or desired result, such as reducing at least one sign orsymptom of an XDH-associated disorder in a subject. Treatment alsoincludes a reduction of one or more sign or symptoms associated withunwanted XDH expression; diminishing the extent of unwanted XDHactivation or stabilization; amelioration or palliation of unwanted XDHactivation or stabilization. “Treatment” can also mean prolongingsurvival as compared to expected survival in the absence of treatment.

The term “lower” in the context of the level of XDH in a subject or adisease marker or symptom refers to a statistically significant decreasein such level. The decrease can be, for example, at least 10%, 15%, 20%,25%, 30%, %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or more. In certain embodiments, a decrease is at least 20%. In certainembodiments, the decrease is at least 50% in a disease marker, e.g.,protein or gene expression level. “Lower” in the context of the level ofXDH in a subject is a decrease to a level accepted as within the rangeof normal for an individual without such disorder. In certainembodiments, the expression of the target is normalized, i.e., decreasedtowards or to a level accepted as within the range of normal for anindividual without such disorder, e.g., normalization of serum uric acidlevel. As used here, “lower” in a subject can refer to lowering of geneexpression or protein production in a cell in a subject does not requirelowering of expression in all cells or tissues of a subject. Forexample, as used herein, lowering in a subject can include lowering ofgene expression or protein production in the liver of a subject.

The term “lower” can also be used in association with normalizing asymptom of a disease or condition, i.e. decreasing the differencebetween a level in a subject suffering from an XDH-associated diseasetowards or to a level in a normal subject not suffering from anXDH-associated disease.

As used herein, if a disease is associated with an elevated value for asymptom, “normal” is considered to be the upper limit of normal. If adisease is associated with a decreased value for a symptom, “normal” isconsidered to be the lower limit of normal.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or condition thereof, that would benefit from areduction in expression of an XDH gene or production of XDH protein,refers to preventing a subject who has at least one sign or symptom of adisease from developing further signs and symptoms thereby meeting thediagnostic criteria for that disease. In certain embodiments, preventionincludes delayed progression to meeting the diagnostic criteria of thedisease by days, weeks, months or years as compared to what would bepredicted by natural history studies or the typical progression of thedisease.

As used herein, the terms “xanthine dehydrogenase-associated disease” or“XDH-associated disease,” include a disease, disorder or condition thatwould benefit from a decrease in XDH gene expression, replication, orprotein activity. Such disorders are caused by, or associated withelevated serum uric acid levels, such as hyperuricemia, includingasymptomatic hyperuricemia.

“Hyperuricemia” is an elevated uric acid level in the blood. The normalupper limit is 6.8 mg/dL, and anything over 7 mg/dL is consideredsaturated, and symptoms can occur. Additional indicators of hyeruricemiainclude, for example, accelerated purine degradation, in high cellturnover states (hemolysis, rhabdomyolysis, and tumor lysis) anddecreased excretion (renal insufficiency and metabolic acidosis).Methods to detect and monitor uric acid in serum or other subjectsamples are known in the art. Uric acid levels can be detected, forexample using carbonate-phosphotungstate method, spectrophotometricuricase method, or chromatotgraphy methods such as HPLC or LCMS.

Hyperuricemia is associated with a number of diseases and conditions,including, but not limited to, gout NAFLD, NASH, metabolic disorder,insulin resistance, cardiovascular disease, hypertension, type 2diabetes, and conditions linked to oxidative stress e.g., chronic lowgrade inflammation; or other XDH-associated disease. Thus, thecompositions and methods of the invention which lower serum urin acidlevels, e.g., lower serum uric acid levels to about to 6.8 mg/dl or less(the level of solubility of uric acid in serum) are also useful to treatsubjects having gout, NAFLD, NASH, metabolic disorder, insulinresistance, cardiovascular disease, hypertension, type 2 diabetes, orconditions linked to oxidative, even in the absence of symptoms

“Gout” is a disorder that allows for the accumulation of uric acid inthe blood and tissues. This leads to the precipitation of uratemonohydrate crystals within a joint. When tissues are saturated withurate, crystals will precipitate. Precipitation is enhanced in acidicenvironments and cold environments, leading to increased precipitationin peripheral joints, such as the great toe. Gout has a malepredominance in a 4:1 ratio of men to women. Uric acid levels can beelevated ten to 15 years before clinical manifestations of gout.

NAFLD is associated with hyperuricemia (Xu et al., J. Hepatol.62:1412-1419, 2015). The diagnosis of“nonalcoholic fatty liver disease”(“NAFLD”) requires that (a) there is evidence of hepatic steatosis,either by imaging or by histology and (b) there are no causes forsecondary hepatic fat accumulation such as significant alcoholconsumption, use of steatogenic medication or hereditary disorders. Inthe majority of patients, NAFLD is associated with metabolic riskfactors such as obesity, diabetes mellitus, and dyslipidemia. NAFLD ishistologically further categorized into “nonalcoholic fatty liver”(“NAFL”) and “nonalcoholic steatohepatitis” (“NASH”). “NAFL” is definedas the presence of hepatic steatosis with no evidence of hepatocellularinjury in the form of ballooning of the hepatocytes. “NASH” is definedas the presence of hepatic steatosis and inflammation with hepatocyteinjury (ballooning) with or without fibrosis (Chalasani et al., Hepatol.55:2005-2023, 2012). It is generally agreed that patients with simplesteatosis have very slow, if any, histological progression, whilepatients with NASH can exhibit histological progression tocirrhotic-stage disease. The long term outcomes of patients with NAFLDand NASH have been reported in several studies. Their findings can besummarized as follows; (a) patients with NAFLD have increased overallmortality compared to matched control populations, (b) the most commoncause of death in patients with NAFLD, NAFL, and NASH is cardiovasculardisease, and (c) patients with NASH (but not NAFL) have an increasedliver-related mortality rate. In a mouse model of NAFLD, treatment withallopurinol both prevented the development of hepatic steatosis, butalso significantly ameliorated established hepatic steatosis in mice (Xuet al., J. Hepatol. 62:1412-1419, 2015).

Cardiovascular disease has been associated with hyperuricemia.Allopurinol has been demonstrated to be effective in the treatment ofcardiovascular disease in animal models and humans including myocardialinfarction, ischemia-reperfusion injury, hypoxia, ischemic heartdisease, heart failure, hypercholesterolemia, and hypertension (Pacheret al., Pharma. Rev. 58:87-114, 2006). As discussed above, treatmentwith allopurinol is contraindicated in a number of populations,especially those with compromised renal function.

Metabolic syndrome, insulin resistance, and type 2 diabetes areassociated with hyperuricemia (Cardoso et al., J. Pediatr. 89:412-418,2013).

“Metabolic syndrome” is characterized by a cluster of conditions definedas at least three of the five following metabolic risk factors whichinclude: large waistline (≥35 inches for women or ≥40 inches for men);high triglyceride level (≥150 mg/dl); low HDL cholesterol (≤50 mg/dl forwomen or ≤40 mg/dl for men); elevated blood pressure (≥130/85) or onmedicine to treat high blood pressure; and high fasting blood sugar(≥100 mg/dl) or being in medicine to treat high blood sugar.

“Insulin resistance” is characterized by the presence of at least oneof: a fasting blood glucose level of 100-125 mg/dL taken at twodifferent times; or an oral glucose tolerance test with a result of aglucose level of 140-199 mg/dL at 2 hours after glucose consumption.

“Type 2 diabetes” is characterized by at least one of: a fasting bloodglucose level ≥126 mg/dL taken at two different times; a hemoglobin A1c(A1C) test with a result of ≥6.5% or higher; or an oral glucosetolerance test with a result of a glucose level ≥200 mg/dL at 2 hoursafter glucose consumption.

Metabolic syndrome, insulin resistance, and type 2 diabetes are oftenassociated with decreased renal function or the potential for decreasedrenal function.

Further details regarding signs and symptoms of the various diseases orconditions are provided herein and are well known in the art.

In one embodiment of the invention, the XDH-associated disease ishyperuricemia.

In another embodiment of the invention, the XDH-associate disease isgout.

In certain embodiments, an XDH-associated disease is NASH or NAFLD.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjecthaving an XDH-associated disease, is sufficient to effect treatment ofthe disease (e.g., by diminishing, ameliorating, or maintaining theexisting disease or one or more symptoms of disease). The“therapeutically effective amount” may vary depending on the RNAi agent,how the agent is administered, the disease and its severity and thehistory, age, weight, family history, genetic makeup, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the subject to be treated. In certain embodiments,treatment with the iRNAs of the invention will result in serum uric acidlevels at 2-6.8 mg/dl, such as, 2-6 mg/dl in subjects. Maintenance ofsuch uric acid levels will treat or prevent XDH-associated diseases.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjecthaving at least one sign or symptom of an XDH-associated disorder, issufficient to prevent or delay the subject's progression to meeting thefull diagnostic criteria of the disease. Prevention of the diseaseincludes slowing the course of progression to full blown disease. The“prophylactically effective amount” may vary depending on the RNAiagent, how the agent is administered, the degree of risk of disease, andthe history, age, weight, family history, genetic makeup, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylactically effectiveamount” also includes an amount of an RNAi agent that produces somedesired effect at a reasonable benefit/risk ratio applicable to anytreatment. The iRNA employed in the methods of the present invention maybe administered in a sufficient amount to produce a reasonablebenefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human subjects and animal subjects without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the subject being treated. Such carriers are knownin the art. Pharmaceutically acceptable carriers include carriers foradministration by injection.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, cerebrospinalfluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samplesmay include samples from tissues, organs, or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In some embodiments, a “sample derived from a subject”refers to urine obtained from the subject. A “sample derived from asubject” can refer to blood or blood derived serum or plasma from thesubject.

II. iRNAs of the Invention

The present invention provides iRNAs which inhibit the expression of axanthine dehydrogenase gene. In some embodiments, the iRNA includesdouble stranded ribonucleic acid (dsRNA) molecules for inhibiting theexpression of an XDH gene in a cell, such as a cell within a subject,e.g., a mammal, such as a human susceptible to developing a xanthinedehydrogenase-associated disorder. The dsRNAi agent includes anantisense strand having a region of complementarity which iscomplementary to at least a part of an mRNA formed in the expression ofan XDH gene. The region of complementarity is about 19-30 nucleotides inlength (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19nucleotides in length). Upon contact with a cell expressing the XDHgene, the iRNA inhibits the expression of the XDH gene (e.g., a human, aprimate, a non-primate, or a rat XDH gene) by at least about 50% asassayed by, for example, a PCR or branched DNA (bDNA)-based method, orby a protein-based method, such as by immunofluorescence analysis,using, for example, western blotting or flow cytometric techniques. Insome embodiments, inhibition of expression is determined by the qPCRmethod provided in the examples herein with the siRNA at, e.g., a 10 nMconcentration, in an appropriate organism cell or cell line providedtherein. In some embodiments, inhibition of expression in vivo isdetermined by knockdown of the human gene in a rodent expressing thehuman gene, e.g., a mouse or an AAV-infected mouse expressing the humantarget gene, e.g., when administered as single dose, e.g., at 3 mg/kg atthe nadir of RNA expression.

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of an XDHgene. The other strand (the sense strand) includes a region that iscomplementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. As described elsewhere herein and as known in the art, thecomplementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is 15 to 30 base pairs in length, e.g.,15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20,15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24,18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25,19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26,20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26,21-25, 21-24, 21-23, or 21-22 base pairs in length. In certainembodiments, the duplex structure is 18 to 25 base pairs in length,e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23,19-22, 19-21, 19-20, 20-25, 20-24,20-23, 20-22, 20-21, 21-25, 21-24,21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24 or 24-25 base pairs inlength, for example, 19-21 basepairs in length. Ranges and lengthsintermediate to the above recited ranges and lengths are alsocontemplated to be part of the disclosure.

Similarly, the region of complementarity to the target sequence is 15 to30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length, for example 19-23 nucleotides in length or 21-23nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of thedisclosure.

In some embodiments, the duplex structure is 19 to 30 base pairs inlength. Similarly, the region of complementarity to the target sequenceis 19 to 30 nucleotides in length.

In some embodiments, the dsRNA is about 19 to about 23 nucleotides inlength, or about 25 to about 30 nucleotides in length. In general, thedsRNA is long enough to serve as a substrate for the Dicer enzyme. Forexample, it is well-known in the art that dsRNAs longer than about 21-23nucleotides in length may serve as substrates for Dicer. As theordinarily skilled person will also recognize, the region of an RNAtargeted for cleavage will most often be part of a larger RNA molecule,often an mRNA molecule. Where relevant, a “part” of an mRNA target is acontiguous sequence of an mRNA target of sufficient length to allow itto be a substrate for RNAi-directed cleavage (i.e., cleavage through aRISC pathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 19to about 30 base pairs, e.g., about 19-30, 19-29, 19-28, 19-27, 19-26,19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27,20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27,21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in oneembodiment, to the extent that it becomes processed to a functionalduplex, of e.g., 15-30 base pairs, that targets a desired RNA forcleavage, an RNA molecule or complex of RNA molecules having a duplexregion greater than 30 base pairs is a dsRNA. Thus, an ordinarilyskilled artisan will recognize that in one embodiment, a miRNA is adsRNA. In another embodiment, a dsRNA is not a naturally occurringmiRNA. In another embodiment, an iRNA agent useful to target xanthinedehydrogenase gene expression is not generated in the target cell bycleavage of a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3,or 4 nucleotides. dsRNAs having at least one nucleotide overhang canhave superior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand, or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end, or both ends of an antisense or sensestrand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art. Doublestranded RNAi compounds of the invention may be prepared using atwo-step procedure. First, the individual strands of the double strandedRNA molecule are prepared separately. Then, the component strands areannealed. The individual strands of the siRNA compound can be preparedusing solution-phase or solid-phase organic synthesis or both. Organicsynthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Similarly, single-stranded oligonucleotides of the invention can beprepared using solution-phase or solid-phase organic synthesis or both.

Regardless of the method of synthesis, the siRNA preparation can beprepared in a solution (e.g., an aqueous or organic solution) that isappropriate for formulation. For example, the siRNA preparation can beprecipitated and redissolved in pure double-distilled water, andlyophilized. The dried siRNA can then be resuspended in a solutionappropriate for the intended formulation process.

In an aspect, a dsRNA of the invention includes at least two nucleotidesequences, a sense sequence and an anti-sense sequence. The sense strandis selected from the group of sequences provided in any one of Tables2-3 and 6-7, and the corresponding antisense strand of the sense strandis selected from the group of sequences of any one of Tables 2-3 and6-7. In this aspect, one of the two sequences is complementary to theother of the two sequences, with one of the sequences beingsubstantially complementary to a sequence of an mRNA generated in theexpression of a xanthine dehydrogenase gene. As such, in this aspect, adsRNA will include two oligonucleotides, where one oligonucleotide isdescribed as the sense strand in any one of Tables 2-3 and 6-7, and thesecond oligonucleotide is described as the corresponding antisensestrand of the sense strand in any one of Tables 2-3 and 6-7.

In certain embodiments, the substantially complementary sequences of thedsRNA are contained on separate oligonucleotides. In other embodiments,the substantially complementary sequences of the dsRNA are contained ona single oligonucleotide.

It will be understood that, although the sequences in Tables 2 and 6 arenot described as modified or conjugated sequences, the RNA of the iRNAof the invention e.g., a dsRNA of the invention, may comprise any one ofthe sequences set forth in any one of Tables 2-3 and 6-7 that isun-modified, un-conjugated, or modified or conjugated differently thandescribed therein. In other words, the invention encompasses dsRNA ofTables 2-3 and 6-7 which are un-modified, un-conjugated, modified, orconjugated, as described herein.

The skilled person is well aware that dsRNAs having a duplex structureof about 20 to 23 base pairs, e.g., 21, base pairs have been hailed asparticularly effective in inducing RNA interference (Elbashir et al.,EMBO 2001, 20:6877-6888). However, others have found that shorter orlonger RNA duplex structures can also be effective (Chu and Rana (2007)RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in any one of Tables 2-3 and 6-7,dsRNAs described herein can include at least one strand of a length ofminimally 21 nucleotides. It can be reasonably expected that shorterduplexes having any one of the sequences in any one of Tables 2-3 and6-7 minus only a few nucleotides on one or both ends can be similarlyeffective as compared to the dsRNAs described above. Hence, dsRNAshaving a sequence of at least 19, 20, or more contiguous nucleotidesderived from any one of the sequences of any one of Tables 2-3 and 6-7,and differing in their ability to inhibit the expression of a xanthinedehydrogenase gene by not more than about 5, 10, 15, 20, 25, or 30%inhibition from a dsRNA comprising the full sequence, are contemplatedto be within the scope of the present invention.

In addition, the RNAs provided in Tables 2-3 and 6-7 identify a site(s)in a xanthine dehydrogenase transcript that is susceptible toRISC-mediated cleavage. As such, the present invention further featuresiRNAs that target within one of these sites. As used herein, an iRNA issaid to target within a particular site of an RNA transcript if the iRNApromotes cleavage of the transcript anywhere within that particularsite. Such an iRNA will generally include at least about 19 contiguousnucleotides from any one of the sequences provided in any one of Tables2-3 and 6-7 coupled to additional nucleotide sequences taken from theregion contiguous to the selected sequence in a xanthine dehydrogenasegene.

An RNAi agent as described herein can contain one or more mismatches tothe target sequence. In one embodiment, an RNAi agent as describedherein contains no more than 3 mismatches (i.e., 3, 2, 1, or 0mismatches). In one embodiment, an RNAi agent as described hereincontains no more than 2 mismatches. In one embodiment, an RNAi agent asdescribed herein contains no more than 1 mismatch. In one embodiment, anRNAi agent as described herein contains 0 mismatches. In certainembodiments, if the antisense strand of the RNAi agent containsmismatches to the target sequence, the mismatch can optionally berestricted to be within the last 5 nucleotides from either the 5′- or3′-end of the region of complementarity. For example, in suchembodiments, for a 23 nucleotide RNAi agent, the strand which iscomplementary to a region of an XDH gene generally does not contain anymismatch within the central 13 nucleotides. The methods described hereinor methods known in the art can be used to determine whether an RNAiagent containing a mismatch to a target sequence is effective ininhibiting the expression of an XDH gene. Consideration of the efficacyof RNAi agents with mismatches in inhibiting expression of an XDH geneis important, especially if the particular region of complementarity inan XDH gene is known to have polymorphic sequence variation within thepopulation.

III. Modified iRNAs of the Invention

In certain embodiments, the RNA of the iRNA of the invention e.g., adsRNA, is un-modified, and does not comprise, e.g., chemicalmodifications or conjugations known in the art and described herein. Inother embodiments, the RNA of an iRNA of the invention, e.g., a dsRNA,is chemically modified to enhance stability or other beneficialcharacteristics. In certain embodiments of the invention, substantiallyall of the nucleotides of an iRNA of the invention are modified. Inother embodiments of the invention, all of the nucleotides of an iRNA orsubstantially all of the nucleotides of an iRNA are modified, i.e., notmore than 5, 4, 3, 2, or 1 unmodified nucleotides are present in astrand of the iRNA.

The nucleic acids featured in the invention can be synthesized ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; or backbonemodifications, including modification or replacement of thephosphodiester linkages. Specific examples of iRNA compounds useful inthe embodiments described herein include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In some embodiments, amodified iRNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′.

Various salts, mixed salts and free acid forms are also included. Insome embodiments of the invention, the dsRNA agents of the invention arein a free acid form. In other embodiments of the invention, the dsRNAagents of the invention are in a salt form. In one embodiment, the dsRNAagents of the invention are in a sodium salt form. In certainembodiments, when the dsRNA agents of the invention are in the sodiumsalt form, sodium ions are present in the agent as counterions forsubstantially all of the phosphodiester or phosphorothiotate groupspresent in the agent. Agents in which substantially all of thephosphodiester or phosphorothioate linkages have a sodium counterioninclude not more than 5, 4, 3, 2, or 1 phosphodiester orphosphorothioate linkages without a sodium counterion. In someembodiments, when the dsRNA agents of the invention are in the sodiumsalt form, sodium ions are present in the agent as counterions for allof the phosphodiester or phosphorothiotate groups present in the agent.

Representative U.S. Patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. No.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S, and CH₂ component parts.

Representative U.S. Patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

Suitable RNA mimetics are contemplated for use in iRNAs provided herein,in which both the sugar and the internucleoside linkage, i.e., thebackbone, of the nucleotide units are replaced with novel groups. Thebase units are maintained for hybridization with an appropriate nucleicacid target compound. One such oligomeric compound in which an RNAmimetic that has been shown to have excellent hybridization propertiesis referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Representative US patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents ofeach of which are hereby incorporated herein by reference. AdditionalPNA compounds suitable for use in the iRNAs of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— of the above-referencedU.S. Pat. No. 5,489,677, and the amide backbones of the above-referencedU.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured hereinhave morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506. The native phosphodiester backbone can be represented asO—P(O))(OH)—OCH2-.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)2. Further exemplary modifications include:5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides,5′-Me-2′-deoxynucleotides, (both R and S isomers in these threefamilies); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative US patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C), anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as deoxythimidine (dT), 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. Patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

An iRNA agent of the disclosure can also be modified to include one ormore bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ringmodified by a ring formed by the bridging of two carbons, whetheradjacent or non-adjacent. A “bicyclic nucleoside” (“BNA”) is anucleoside having a sugar moiety comprising a ring formed by bridgingtwo carbons, whether adjacent or non-adjacent, of the sugar ring,thereby forming a bicyclic ring system. In certain embodiments, thebridge connects the 4′-carbon and the 2′-carbon of the sugar ring,optionally, via the 2′-acyclic oxygen atom. Thus, in some embodiments anagent of the invention may include one or more locked nucleic acids(LNA). A locked nucleic acid is a nucleotide having a modified ribosemoiety in which the ribose moiety comprises an extra bridge connectingthe 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprisinga bicyclic sugar moiety comprising a 4′-CH₂—O-2′ bridge. This structureeffectively “locks” the ribose in the 3′-endo structural conformation.The addition of locked nucleic acids to siRNAs has been shown toincrease siRNA stability in serum, and to reduce off-target effects(Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al.,(2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclicnucleosides for use in the polynucleotides of the invention includewithout limitation nucleosides comprising a bridge between the 4′ andthe 2′ ribosyl ring atoms. In certain embodiments, the antisensepolynucleotide agents of the invention include one or more bicyclicnucleosides comprising a 4′ to 2′ bridge.

A locked nucleoside can be represented by the structure (omittinstereochemistry),

wherein B is a nucleobase or modified nucleobase and L is the linkinggroup that joins the 2′-carbon to the 4′-carbon of the ribose ring.

Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but arenot limited to 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA);4′-CH(CH₃)—O-2′ (also referred to as “constrained ethyl” or “cEt”) and4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof; see, e.g., U.S. Pat. No.7,399,845); 4′-C(CH₃(CH₃)—O-2′ (and analogs thereof; see e.g., U.S. Pat.No. 8,278,283); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof; see e.g., U.S.Pat. No. 8,278,425); 4′-CH₂—O—N(CH₃)-2′ (see, e.g., U.S. PatentPublication No. 2004/0171570); 4′-CH₂—N(R)—O-2′, wherein R is H, C1-C12alkyl, or a nitrogen protecting group (see, e.g., U.S. Pat. No.7,427,672); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya et al., J.Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogsthereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents ofeach of the foregoing are hereby incorporated herein by reference.

Additional representative U.S. Patents and U.S. Patent Publications thatteach the preparation of locked nucleic acid nucleotides include, butare not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191;6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193;8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or moreconstrained ethyl nucleotides. As used herein, a “constrained ethylnucleotide” or “cEt” is a locked nucleic acid comprising a bicyclicsugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge (i.e., L in thepreceding structure). In one embodiment, a constrained ethyl nucleotideis in the S conformation referred to herein as “S-cEt.”

An iRNA of the invention may also include one or more “conformationallyrestricted nucleotides” (“CRN”). CRN are nucleotide analogs with alinker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′carbons of ribose. CRN lock the ribose ring into a stable conformationand increase the hybridization affinity to mRNA. The linker is ofsufficient length to place the oxygen in an optimal position forstability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of theabove noted CRN include, but are not limited to, US2013/0190383; andWO2013/036868, the entire contents of each of which are herebyincorporated herein by reference.

In some embodiments, an iRNA of the invention comprises one or moremonomers that are UNA (unlocked nucleic acid) nucleotides. UNA isunlocked acyclic nucleic acid, wherein any of the bonds of the sugar hasbeen removed, forming an unlocked “sugar” residue. In one example, UNAalso encompasses monomer with bonds between C1′-C4′ have been removed(i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′carbons). In another example, the C2′-C3′ bond (i.e. the covalentcarbon-carbon bond between the C2′ and C3′ carbons) of the sugar hasbeen removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) andFluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated byreference).

Representative U.S. publications that teach the preparation of UNAinclude, but are not limited to, U.S. Pat. No. 8,314,227; andUS2013/0096289; US2013/0011922; and US2011/0313020, the entire contentsof each of which are hereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in WO2011/005861.

Other modifications of the nucleotides of an iRNA of the inventioninclude a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminalphosphate or phosphate mimic on the antisense strand of an iRNA.Suitable phosphate mimics are disclosed in, for example US2012/0157511,the entire contents of which are incorporated herein by reference.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double stranded RNA agents ofthe invention include agents with chemical modifications as disclosed,for example, in WO2013/075035, the entire contents of each of which areincorporated herein by reference. As shown herein and in WO2013/075035,one or more motifs of three identical modifications on three consecutivenucleotides may be introduced into a sense strand or antisense strand ofa dsRNAi agent, particularly at or near the cleavage site. In someembodiments, the sense strand and antisense strand of the dsRNAi agentmay otherwise be completely modified. The introduction of these motifsinterrupts the modification pattern, if present, of the sense orantisense strand. The dsRNAi agent may be optionally conjugated with aGalNAc derivative ligand, for instance on the sense strand.

More specifically, when the sense strand and antisense strand of thedouble stranded RNA agent are completely modified to have one or moremotifs of three identical modifications on three consecutive nucleotidesat or near the cleavage site of at least one strand of a dsRNAi agent,the gene silencing activity of the dsRNAi agent was observed.

Accordingly, the invention provides double stranded RNA agents capableof inhibiting the expression of a target gene (i.e., XDH gene) in vivo.The RNAi agent comprises a sense strand and an antisense strand. Eachstrand of the RNAi agent may be, for example, 17-30 nucleotides inlength, 25-30 nucleotides in length, 27-30 nucleotides in length, 19-25nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides inlength, 21-25 nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex doublestranded RNA (“dsRNA”), also referred to herein as “dsRNAi agent.” Theduplex region of a dsRNAi agent may be, for example, the duplex regioncan be 27-30 nucleotide pairs in length, 19-25 nucleotide pairs inlength, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs inlength, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs inlength. In another example, the duplex region is selected from 19, 20,21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In certain embodiments, the dsRNAi agent may contain one or moreoverhang regions or capping groups at the 3′-end, 5′-end, or both endsof one or both strands. The overhang can be, independently, 1-6nucleotides in length, for instance 2-6 nucleotides in length, 1-5nucleotides in length, 2-5 nucleotides in length, 14 nucleotides inlength, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3nucleotides in length, or 1-2 nucleotides in length. In certainembodiments, the overhang regions can include extended overhang regionsas provided above. The overhangs can be the result of one strand beinglonger than the other, or the result of two strands of the same lengthbeing staggered. The overhang can form a mismatch with the target mRNAor it can be complementary to the gene sequences being targeted or canbe another sequence. The first and second strands can also be joined,e.g., by additional bases to form a hairpin, or by other non-baselinkers.

In certain embodiments, the nucleotides in the overhang region of thedsRNAi agent can each independently be a modified or unmodifiednucleotide including, but no limited to 2′-sugar modified, such as,2′-F, 2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine(Teo), 2′-O-methoxyethyladenosine (Aeo),2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinationsthereof.

For example, TT can be an overhang sequence for either end on eitherstrand. The overhang can form a mismatch with the target mRNA or it canbe complementary to the gene sequences being targeted or can be anothersequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand, or bothstrands of the dsRNAi agent may be phosphorylated. In some embodiments,the overhang region(s) contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In some embodiments, the overhang ispresent at the 3′-end of the sense strand, antisense strand, or bothstrands. In some embodiments, this 3′-overhang is present in theantisense strand. In some embodiments, this 3′-overhang is present inthe sense strand.

The dsRNAi agent may contain only a single overhang, which canstrengthen the interference activity of the RNAi, without affecting itsoverall stability. For example, the single-stranded overhang may belocated at the 3′-end of the sense strand or, alternatively, at the3′-end of the antisense strand. The RNAi may also have a blunt end,located at the 5′-end of the antisense strand (or the 3′-end of thesense strand) or vice versa. Generally, the antisense strand of thedsRNAi agent has a nucleotide overhang at the 3′-end, and the 5′-end isblunt. While not wishing to be bound by theory, the asymmetric blunt endat the 5′-end of the antisense strand and 3′-end overhang of theantisense strand favor the guide strand loading into RISC process.

In certain embodiments, the dsRNAi agent is a double blunt-ended of 19nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 7, 8, and 9 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, and 13 from the 5′end.

In other embodiments, the dsRNAi agent is a double blunt-ended of 20nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 8, 9, and 10 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, and 13 from the 5′end.

In yet other embodiments, the dsRNAi agent is a double blunt-ended of 21nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 9, 10, and 11 from the 5′end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, and 13 from the 5′end.

In certain embodiments, the dsRNAi agent comprises a 21 nucleotide sensestrand and a 23 nucleotide antisense strand, wherein the sense strandcontains at least one motif of three 2′-F modifications on threeconsecutive nucleotides at positions 9, 10, and 11 from the 5′end; theantisense strand contains at least one motif of three 2′-O-methylmodifications on three consecutive nucleotides at positions 11, 12, and13 from the 5′end, wherein one end of the RNAi agent is blunt, while theother end comprises a 2 nucleotide overhang. In some embodiments, the 2nucleotide overhang is at the 3′-end of the antisense strand.

When the 2 nucleotide overhang is at the 3′-end of the antisense strand,there may be two phosphorothioate internucleotide linkages between theterminal three nucleotides, wherein two of the three nucleotides are theoverhang nucleotides, and the third nucleotide is a paired nucleotidenext to the overhang nucleotide. In one embodiment, the RNAi agentadditionally has two phosphorothioate internucleotide linkages betweenthe terminal three nucleotides at both the 5′-end of the sense strandand at the 5′-end of the antisense strand. In certain embodiments, everynucleotide in the sense strand and the antisense strand of the dsRNAiagent, including the nucleotides that are part of the motifs aremodified nucleotides. In certain embodiments each residue isindependently modified with a 2′-O-methyl or 3′-fluoro, e.g., in analternating motif. Optionally, the dsRNAi agent further comprises aligand (such as, GalNAc).

In certain embodiments, the dsRNAi agent comprises a sense and anantisense strand, wherein the sense strand is 25-30 nucleotide residuesin length, wherein starting from the 5′ terminal nucleotide (position 1)positions 1 to 23 of the first strand comprise at least 8ribonucleotides; the antisense strand is 36-66 nucleotide residues inlength and, starting from the 3′ terminal nucleotide, comprises at least8 ribonucleotides in the positions paired with positions 1-23 of sensestrand to form a duplex; wherein at least the 3′ terminal nucleotide ofantisense strand is unpaired with sense strand, and up to 6 consecutive3′ terminal nucleotides are unpaired with sense strand, thereby forminga 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′terminus of antisense strand comprises from 10-30 consecutivenucleotides which are unpaired with sense strand, thereby forming a10-30 nucleotide single stranded 5′ overhang; wherein at least the sensestrand 5′ terminal and 3′ terminal nucleotides are base paired withnucleotides of antisense strand when sense and antisense strands arealigned for maximum complementarity, thereby forming a substantiallyduplexed region between sense and antisense strands; and antisensestrand is sufficiently complementary to a target RNA along at least 19ribonucleotides of antisense strand length to reduce target geneexpression when the double stranded nucleic acid is introduced into amammalian cell; and wherein the sense strand contains at least one motifof three 2′-F modifications on three consecutive nucleotides, where atleast one of the motifs occurs at or near the cleavage site. Theantisense strand contains at least one motif of three 2′-O-methylmodifications on three consecutive nucleotides at or near the cleavagesite.

In certain embodiments, the dsRNAi agent comprises sense and antisensestrands, wherein the dsRNAi agent comprises a first strand having alength which is at least 25 and at most 29 nucleotides and a secondstrand having a length which is at most 30 nucleotides with at least onemotif of three 2′-O-methyl modifications on three consecutivenucleotides at position 11, 12, and 13 from the 5′ end; wherein the 3′end of the first strand and the 5′ end of the second strand form a bluntend and the second strand is 1-4 nucleotides longer at its 3′ end thanthe first strand, wherein the duplex region which is at least 25nucleotides in length, and the second strand is sufficientlycomplementary to a target mRNA along at least 19 nucleotide of thesecond strand length to reduce target gene expression when the RNAiagent is introduced into a mammalian cell, and wherein Dicer cleavage ofthe dsRNAi agent results in an siRNA comprising the 3′-end of the secondstrand, thereby reducing expression of the target gene in the mammal.Optionally, the dsRNAi agent further comprises a ligand.

In certain embodiments, the sense strand of the dsRNAi agent contains atleast one motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In certain embodiments, the antisense strand of the dsRNAi agent canalso contain at least one motif of three identical modifications onthree consecutive nucleotides, where one of the motifs occurs at or nearthe cleavage site in the antisense strand.

For a dsRNAi agent having a duplex region of 19-23 nucleotides inlength, the cleavage site of the antisense strand is typically aroundthe 10, 11, and 12 positions from the 5′-end. Thus the motifs of threeidentical modifications may occur at the 9, 10, and 11 positions; the10, 11, and 12 positions; the 11, 12, and 13 positions; the 12, 13, and14 positions; or the 13, 14, and 15 positions of the antisense strand,the count starting from the first nucleotide from the 5′-end of theantisense strand, or, the count starting from the first pairednucleotide within the duplex region from the 5′-end of the antisensestrand. The cleavage site in the antisense strand may also changeaccording to the length of the duplex region of the dsRNAi agent fromthe 5′-end.

The sense strand of the dsRNAi agent may contain at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesmay overlap, or all three nucleotides may overlap.

In some embodiments, the sense strand of the dsRNAi agent may containmore than one motif of three identical modifications on threeconsecutive nucleotides. The first motif may occur at or near thecleavage site of the strand and the other motifs may be a wingmodification. The term “wing modification” herein refers to a motifoccurring at another portion of the strand that is separated from themotif at or near the cleavage site of the same strand. The wingmodification is either adjacent to the first motif or is separated by atleast one or more nucleotides. When the motifs are immediately adjacentto each other then the chemistries of the motifs are distinct from eachother, and when the motifs are separated by one or more nucleotide thanthe chemistries can be the same or different. Two or more wingmodifications may be present. For instance, when two wing modificationsare present, each wing modification may occur at one end relative to thefirst motif which is at or near cleavage site or on either side of thelead motif.

Like the sense strand, the antisense strand of the dsRNAi agent maycontain more than one motifs of three identical modifications on threeconsecutive nucleotides, with at least one of the motifs occurring at ornear the cleavage site of the strand. This antisense strand may alsocontain one or more wing modifications in an alignment similar to thewing modifications that may be present on the sense strand.

In some embodiments, the wing modification on the sense strand orantisense strand of the dsRNAi agent typically does not include thefirst one or two terminal nucleotides at the 3′-end, 5′-end, or bothends of the strand.

In other embodiments, the wing modification on the sense strand orantisense strand of the dsRNAi agent typically does not include thefirst one or two paired nucleotides within the duplex region at the3′-end, 5′-end, or both ends of the strand.

When the sense strand and the antisense strand of the dsRNAi agent eachcontain at least one wing modification, the wing modifications may fallon the same end of the duplex region, and have an overlap of one, two,or three nucleotides.

When the sense strand and the antisense strand of the dsRNAi agent eachcontain at least two wing modifications, the sense strand and theantisense strand can be so aligned that two modifications each from onestrand fall on one end of the duplex region, having an overlap of one,two, or three nucleotides; two modifications each from one strand fallon the other end of the duplex region, having an overlap of one, two orthree nucleotides; two modifications one strand fall on each side of thelead motif, having an overlap of one, two or three nucleotides in theduplex region.

In some embodiments, every nucleotide in the sense strand and antisensestrand of the dsRNAi agent, including the nucleotides that are part ofthe motifs, may be modified. Each nucleotide may be modified with thesame or different modification which can include one or more alterationof one or both of the non-linking phosphate oxygens or of one or more ofthe linking phosphate oxygens; alteration of a constituent of the ribosesugar, e.g., of the 2′-hydroxyl on the ribose sugar; wholesalereplacement of the phosphate moiety with “dephospho” linkers;modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking O of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′- or 5′ terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of an RNA or may only occur in a single strand region of aRNA. For example, a phosphorothioate modification at a non-linking Oposition may only occur at one or both termini, may only occur in aterminal region, e.g., at a position on a terminal nucleotide or in thelast 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in doublestrand and single strand regions, particularly at termini. The 5′-end orends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′- or 3′-overhang,or in both. For example, it can be desirable to include purinenucleotides in overhangs. In some embodiments all or some of the basesin a 3′- or 5′-overhang may be modified, e.g., with a modificationdescribed herein. Modifications can include, e.g., the use ofmodifications at the 2′ position of the ribose sugar with modificationsthat are known in the art, e.g., the use of deoxyribonucleotides,2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of theribosugar of the nucleobase, and modifications in the phosphate group,e.g., phosphorothioate modifications. Overhangs need not be homologouswith the target sequence.

In some embodiments, each residue of the sense strand and antisensestrand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C— allyl, 2′-deoxy,2′-hydroxyl, or 2′-fluoro. The strands can contain more than onemodification. In one embodiment, each residue of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-O-methyl or 2′-fluoro modifications, or others.

In certain embodiments, the N_(a) or N_(b) comprise modifications of analternating pattern. The term “alternating motif” as used herein refersto a motif having one or more modifications, each modification occurringon alternating nucleotides of one strand. The alternating nucleotide mayrefer to one per every other nucleotide or one per every threenucleotides, or a similar pattern. For example, if A, B and C eachrepresent one type of modification to the nucleotide, the alternatingmotif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB. . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC. . . ,” etc.

The type of modifications contained in the alternating motif may be thesame or different.

For example, if A, B, C, D each represent one type of modification onthe nucleotide, the alternating pattern, i.e., modifications on everyother nucleotide, may be the same, but each of the sense strand orantisense strand can be selected from several possibilities ofmodifications within the alternating motif such as “ABABAB . . . ”,“ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.

In some embodiments, the dsRNAi agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′ to 3′ of the strand and the alternating motif inthe antisense strand may start with “BABABA” from 5′ to 3′ of the strandwithin the duplex region. As another example, the alternating motif inthe sense strand may start with “AABBAABB” from 5′ to 3′ of the strandand the alternating motif in the antisense strand may start with“BBAABBAA” from 5′ to 3′ of the strand within the duplex region, so thatthere is a complete or partial shift of the modification patternsbetween the sense strand and the antisense strand.

In some embodiments, the dsRNAi agent comprises the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe sense strand initially has a shift relative to the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe antisense strand initially, i.e., the 2′-O-methyl modifiednucleotide on the sense strand base pairs with a 2′-F modifiednucleotide on the antisense strand and vice versa. The 1 position of thesense strand may start with the 2′-F modification, and the 1 position ofthe antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modificationson three consecutive nucleotides to the sense strand or antisense strandinterrupts the initial modification pattern present in the sense strandor antisense strand. This interruption of the modification pattern ofthe sense or antisense strand by introducing one or more motifs of threeidentical modifications on three consecutive nucleotides to the sense orantisense strand may enhance the gene silencing activity against thetarget gene.

In some embodiments, when the motif of three identical modifications onthree consecutive nucleotides is introduced to any of the strands, themodification of the nucleotide next to the motif is a differentmodification than the modification of the motif. For example, theportion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) .. . ,” where “Y” represents the modification of the motif of threeidentical modifications on three consecutive nucleotide, and “N_(a)” and“N_(b)” represent a modification to the nucleotide next to the motif“YYY” that is different than the modification of Y, and where N_(a) andN_(b) can be the same or different modifications. Alternatively, N_(a)or N_(b) may be present or absent when there is a wing modificationpresent.

The iRNA may further comprise at least one phosphorothioate ormethylphosphonate internucleotide linkage. The phosphorothioate ormethylphosphonate internucleotide linkage modification may occur on anynucleotide of the sense strand, antisense strand, or both strands in anyposition of the strand. For instance, the internucleotide linkagemodification may occur on every nucleotide on the sense strand orantisense strand; each internucleotide linkage modification may occur inan alternating pattern on the sense strand or antisense strand; or thesense strand or antisense strand may contain both internucleotidelinkage modifications in an alternating pattern. The alternating patternof the internucleotide linkage modification on the sense strand may bethe same or different from the antisense strand, and the alternatingpattern of the internucleotide linkage modification on the sense strandmay have a shift relative to the alternating pattern of theinternucleotide linkage modification on the antisense strand. In oneembodiment, a double-stranded RNAi agent comprises 6-8 phosphorothioateinternucleotide linkages. In some embodiments, the antisense strandcomprises two phosphorothioate internucleotide linkages at the 5′-endand two phosphorothioate internucleotide linkages at the 3′-end, and thesense strand comprises at least two phosphorothioate internucleotidelinkages at either the 5′-end or the 3′-end.

In some embodiments, the dsRNAi agent comprises a phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region may contain two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within the duplex region. For example, at least 2, 3, 4, orall the overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. These terminal three nucleotides may be at the 3′-end of theantisense strand, the 3′-end of the sense strand, the 5′-end of theantisense strand, or the 5′end of the antisense strand.

In some embodiments, the 2-nucleotide overhang is at the 3′-end of theantisense strand, and there are two phosphorothioate internucleotidelinkages between the terminal three nucleotides, wherein two of thethree nucleotides are the overhang nucleotides, and the third nucleotideis a paired nucleotide next to the overhang nucleotide. Optionally, thedsRNAi agent may additionally have two phosphorothioate internucleotidelinkages between the terminal three nucleotides at both the 5′-end ofthe sense strand and at the 5′-end of the antisense strand.

In one embodiment, the dsRNAi agent comprises mismatch(es) with thetarget, within the duplex, or combinations thereof. The mismatch mayoccur in the overhang region or the duplex region. The base pair may beranked on the basis of their propensity to promote dissociation ormelting (e.g., on the free energy of association or dissociation of aparticular pairing, the simplest approach is to examine the pairs on anindividual pair basis, though next neighbor or similar analysis can alsobe used). In terms of promoting dissociation: A:U is preferred over G:C;G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine).Mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings; and pairings which include a universal base are preferred overcanonical pairings.

In certain embodiments, the dsRNAi agent comprises at least one of thefirst 1, 2, 3, 4, or 5 base pairs within the duplex regions from the5′-end of the antisense strand independently selected from the group of:A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other thancanonical pairings or pairings which include a universal base, topromote the dissociation of the antisense strand at the 5′-end of theduplex.

In certain embodiments, the nucleotide at the 1 position within theduplex region from the 5′-end in the antisense strand is selected fromA, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or3 base pair within the duplex region from the 5′-end of the antisensestrand is an AU base pair. For example, the first base pair within theduplex region from the 5′-end of the antisense strand is an AU basepair.

In other embodiments, the nucleotide at the 3′-end of the sense strandis deoxythimidine (dT) or the nucleotide at the 3′-end of the antisensestrand is deoxythimidine (dT). For example, there is a short sequence ofdeoxythimidine nucleotides, for example, two dT nucleotides on the3′-end of the sense, antisense strand, or both strands.

In certain embodiments, the sense strand sequence may be represented byformula (I):

5′n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)-N_(a)-n_(q)3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY, and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. In someembodiments, YYY is all 2′-F modified nucleotides.

In some embodiments, the N_(a) or N_(b) comprises modifications ofalternating pattern.

In some embodiments, the YYY motif occurs at or near the cleavage siteof the sense strand. For example, when the dsRNAi agent has a duplexregion of 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8; 7,8, 9; 8, 9, 10; 9, 10, 11; 10, 11,12; or 11, 12, 13) of the sensestrand, the count starting from the first nucleotide, from the 5′-end;or optionally, the count starting at the first paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:

5′n_(p)-N_(a)—YYY—N_(b)—ZZZ—N_(a)-n_(q)3′  (Ib);

5′n_(p)-N_(a)—XXX—N_(b)—YYY—N_(a)-n_(q)3′  (Ic); or

5′n_(p)-N_(a)—XXX—N_(b)—YYY—N_(b)—ZZZ—N_(a)-n_(q)3′  (Id).

When the sense strand is represented by formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 04, 0-2, or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 04,0-2, or 0 modified nucleotides. Each N_(a) can independently representan oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Id), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. In some embodiments,N_(b) is 0, 1, 2, 3, 4, 5, or 6. Each N_(a) can independently representan oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may berepresented by the formula:

5′n_(p)-N_(a)—YYY—N_(a)-n_(q)3′  (Ia).

When the sense strand is represented by formula (Ia), each N_(a)independently can represent an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may berepresented by formula (II):

5′n_(q′)—N_(a)′—(Z′Z′Z′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(X′X′X′)_(l)—N′_(a)-n_(p)′3′  (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In some embodiments, the N_(a)′ or N_(b)′ comprises modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the dsRNAi agent has a duplex region of 17-23nucleotides in length, the Y′Y′Y′ motif can occur at positions 9, 10,11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisensestrand, with the count starting from the first nucleotide, from the5′-end; or optionally, the count starting at the first paired nucleotidewithin the duplex region, from the 5′-end. In some embodiments, theY′Y′Y′ motif occurs at positions 11, 12, 13.

In certain embodiments, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In certain embodiments, k is 1 and 1 is 0, or k is 0 and 1 is 1, or bothk and l are 1.

The antisense strand can therefore be represented by the followingformulas:

5′n_(q′)—N_(a)′—Z′Z′Z′—N_(b)′—Y′Y′Y′—N_(a)′-n_(p′)3′  (IIb);

5′n_(q′)—N_(a)′—Y′Y′Y′—N_(b)′—X′X′X′-n_(p)-3′  (IIc); or

5′n_(q′)—N_(a)′—Z′Z′Z′—N_(b)′—Y′Y′Y′—N_(b)′—X′X′X′—N_(a)′-n_(p′)3′  (IId).

When the antisense strand is represented by formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 04, 0-2, or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 04, 0-2, or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. In some embodiments, N_(b) is 0, 1,2, 3, 4, 5, or 6.

In other embodiments, k is 0 and 1 is 0 and the antisense strand may berepresented by the formula:

5′n_(p′)—N_(a′)—Y′Y′Y′—N_(a′)-n_(q′)3′  (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, CRN, UNA, cEt, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C— allyl, 2′-hydroxyl, or2′-fluoro. For example, each nucleotide of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro. Each X, Y, Z, X′, Y′, and Z′, in particular, may represent a2′-O-methyl modification or a 2′-fluoro modification.

In some embodiments, the sense strand of the dsRNAi agent may containYYY motif occurring at 9, 10, and 11 positions of the strand when theduplex region is 21 nt, the count starting from the first nucleotidefrom the 5′-end, or optionally, the count starting at the first pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification. The sense strand may additionally contain XXX motifor ZZZ motifs as wing modifications at the opposite end of the duplexregion; and XXX and ZZZ each independently represents a 2′-OMemodification or 2′-F modification.

In some embodiments the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe first nucleotide from the 5′-end, or optionally, the count startingat the first paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification.

The sense strand represented by any one of the above formulas (Ia),(Ib), (Ic), and (Id) forms a duplex with an antisense strand beingrepresented by any one of formulas (IIa), (IIb), (IIc), and (IId),respectively.

Accordingly, the dsRNAi agents for use in the methods of the inventionmay comprise a sense strand and an antisense strand, each strand having14 to 30 nucleotides, the iRNA duplex represented by formula (III):

sense: 5′n_(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n_(q)3′

antisense:3′n_(p)′—N_(a)′—(X′X′X′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(Z′Z′Z′)_(l)—N_(a)′-n_(q)′5′  (III)

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or maynot be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0and j is 1; or both i and j are 0; or both i and j are 1. In anotherembodiment, k is 0 and 1 is 0; or k is 1 and 1 is 0; k is 0 and 1 is 1;or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand formingan iRNA duplex include the formulas below:

5′n_(p)-N_(a)—YYY—N_(a)-n_(q)3′

3′n_(p)′—N_(a)′—Y′Y′Y′—N_(a)′n_(q)′5′  (IIIa)

5′n_(p)-N_(a)—YYY—N_(b)—ZZZ—N_(a)-n_(q)3′

3′n_(p)′—N_(a)′—Y′Y′Y′—N_(b)′—Z′Z′Z′—N_(a)′n_(q)′5′  (IIIb)

5′n_(p)-N_(a)—XXX—N_(b)—YYY-N_(a)-n_(q)3′

3′n_(p)′—N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(a)′-n_(q)′5′  (IIIc)

5′n_(p)-N_(a)—XXX—N_(b)—YYY—N_(b)—ZZZ—N_(a)-n_(q)3′

3′n_(p)′—N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(b)′—Z′Z′Z′—N_(a)-n_(q)′5′  (IIId)

When the dsRNAi agent is represented by formula (IIIa), each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented by formula (IIIb), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5, or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the dsRNAi agent is represented as formula (IIIc), each N_(b),N_(b)′ independently represents an oligonucleotide sequence comprising0-10, 0-7, 0-10, 0-7, 0-5, 04, 0-2, or 0 modified nucleotides. EachN_(a) independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented as formula (IIId), each N_(b),N_(b)′ independently represents an oligonucleotide sequence comprising0-10, 0-7, 0-10, 0-7, 0-5, 04, 0-2, or 0 modified nucleotides. EachN_(a), N_(a)′ independently represents an oligonucleotide sequencecomprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a),N_(a)′, N_(b,) and N_(b)′ independently comprises modifications ofalternating pattern.

Each of X, Y, and Z in formulas (III), (IIIa), (IIIb), (IIIc), and(IIId) may be the same or different from each other.

When the dsRNAi agent is represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), at least one of the Y nucleotides may form a basepair with one of the Y′ nucleotides. Alternatively, at least two of theY nucleotides form base pairs with the corresponding Y′ nucleotides; orall three of the Y nucleotides all form base pairs with thecorresponding Y′ nucleotides.

When the dsRNAi agent is represented by formula (IIIb) or (IIId), atleast one of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the dsRNAi agent is represented as formula (IIIc) or (IIId), atleast one of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In certain embodiments, the modification on the Y nucleotide isdifferent than the modification on the Y′ nucleotide, the modificationon the Z nucleotide is different than the modification on the Z′nucleotide, or the modification on the X nucleotide is different thanthe modification on the X′ nucleotide.

In certain embodiments, when the dsRNAi agent is represented by formula(IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications. In other embodiments, when the RNAi agent is representedby formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications and n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide a via phosphorothioate linkage. In yet otherembodiments, when the RNAi agent is represented by formula (IIId), theN_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0and at least one n_(p)′ is linked to a neighboring nucleotide viaphosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker (described below). In other embodiments, when the RNAiagent is represented by formula (IIId), the N_(a) modifications are2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′is linked to a neighboring nucleotide via phosphorothioate linkage, thesense strand comprises at least one phosphorothioate linkage, and thesense strand is conjugated to one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker.

In some embodiments, when the dsRNAi agent is represented by formula(IIIa), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications, n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide via phosphorothioate linkage, the sense strandcomprises at least one phosphorothioate linkage, and the sense strand isconjugated to one or more GalNAc derivatives attached through a bivalentor trivalent branched linker.

In some embodiments, the dsRNAi agent is a multimer containing at leasttwo duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and(IIId), wherein the duplexes are connected by a linker. The linker canbe cleavable or non-cleavable. Optionally, the multimer furthercomprises a ligand. Each of the duplexes can target the same gene or twodifferent genes; or each of the duplexes can target same gene at twodifferent target sites.

In some embodiments, the dsRNAi agent is a multimer containing three,four, five, six, or more duplexes represented by formula (III), (IIIa),(IIIb), (IIIc), and (IIId), wherein the duplexes are connected by alinker. The linker can be cleavable or non-cleavable. Optionally, themultimer further comprises a ligand. Each of the duplexes can target thesame gene or two different genes; or each of the duplexes can targetsame gene at two different target sites.

In one embodiment, two dsRNAi agents represented by at least one offormulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to eachother at the 5′ end, and one or both of the 3′ ends, and are optionallyconjugated to a ligand. Each of the agents can target the same gene ortwo different genes; or each of the agents can target same gene at twodifferent target sites.

In certain embodiments, an RNAi agent of the invention may contain a lownumber of nucleotides containing a 2′-fluoro modification, e.g., 10 orfewer nucleotides with 2′-fluoro modification. For example, the RNAiagent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 nucleotides with a2′-fluoro modification. In a specific embodiment, the RNAi agent of theinvention contains 10 nucleotides with a 2′-fluoro modification, e.g., 4nucleotides with a 2′-fluoro modification in the sense strand and 6nucleotides with a 2′-fluoro modification in the antisense strand. Inanother specific embodiment, the RNAi agent of the invention contains 6nucleotides with a 2′-fluoro modification, e.g., 4 nucleotides with a2′-fluoro modification in the sense strand and 2 nucleotides with a2′-fluoro modification in the antisense strand.

In other embodiments, an RNAi agent of the invention may contain anultra low number of nucleotides containing a 2′-fluoro modification,e.g., 2 or fewer nucleotides containing a 2′-fluoro modification. Forexample, the RNAi agent may contain 2, 1 of 0 nucleotides with a2′-fluoro modification. In a specific embodiment, the RNAi agent maycontain 2 nucleotides with a 2′-fluoro modification, e.g., 0 nucleotideswith a 2-fluoro modification in the sense strand and 2 nucleotides witha 2′-fluoro modification in the antisense strand.

Various publications describe multimeric iRNAs that can be used in themethods of the invention. Such publications include WO2007/091269, U.S.Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887, andWO2011/031520 the entire contents of each of which are herebyincorporated herein by reference.

In certain embodiments, the compositions and methods of the disclosureinclude a vinyl phosphonate (VP) modification of an RNAi agent asdescribed herein. In exemplary embodiments, a 5′-vinyl phosphonatemodified nucleotide of the disclosure has the structure:

wherein X is O or S;

R is hydrogen, hydroxy, fluoro, or C₁₋₂₀alkoxy (e.g., methoxy orn-hexadecyloxy);

R^(5′) is ═C(H)—P(O)(OH)₂ and the double bond between the C5′ carbon andR^(5′) is in the E or Z orientation (e.g., E orientation); and

B is a nucleobase or a modified nucleobase, optionally where B isadenine, guanine, cytosine, thymine, or uracil.

A vinyl phosphonate of the instant disclosure may be attached to eitherthe antisense or the sense strand of a dsRNA of the disclosure. Incertain embodiments, a vinyl phosphonate of the instant disclosure isattached to the antisense strand of a dsRNA, optionally at the 5′ end ofthe antisense strand of the dsRNA.

Vinyl phosphonate modifications are also contemplated for thecompositions and methods of the instant disclosure. An exemplary vinylphosphonate structure includes the preceding structure, where R5′ is═C(H)—OP(O)(OH)2 and the double bond between the C5′ carbon and R5′ isin the E or Z orientation (e.g., E orientation).

As described in more detail below, the iRNA that contains conjugationsof one or more carbohydrate moieties to an iRNA can optimize one or moreproperties of the iRNA. In many cases, the carbohydrate moiety will beattached to a modified subunit of the iRNA. For example, the ribosesugar of one or more ribonucleotide subunits of an iRNA can be replacedwith another moiety, e.g., a non-carbohydrate (such as, cyclic) carrierto which is attached a carbohydrate ligand. A ribonucleotide subunit inwhich the ribose sugar of the subunit has been so replaced is referredto herein as a ribose replacement modification subunit (RRMS). A cycliccarrier may be a carbocyclic ring system, i.e., all ring atoms arecarbon atoms, or a heterocyclic ring system, i.e., one or more ringatoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cycliccarrier may be a monocyclic ring system, or may contain two or morerings, e.g. fused rings. The cyclic carrier may be a fully saturatedring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,” such as,two “backbone attachment points” and (ii) at least one “tetheringattachment point.” A “backbone attachment point” as used herein refersto a functional group, e.g. a hydroxyl group, or generally, a bondavailable for, and that is suitable for incorporation of the carrierinto the backbone, e.g., the phosphate, or modified phosphate, e.g.,sulfur containing, backbone, of a ribonucleic acid. A “tetheringattachment point” (TAP) in some embodiments refers to a constituent ringatom of the cyclic carrier, e.g., a carbon atom or a heteroatom(distinct from an atom which provides a backbone attachment point), thatconnects a selected moiety. The moiety can be, e.g., a carbohydrate,e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide,oligosaccharide, or polysaccharide. Optionally, the selected moiety isconnected by an intervening tether to the cyclic carrier. Thus, thecyclic carrier will often include a functional group, e.g., an aminogroup, or generally, provide a bond, that is suitable for incorporationor tethering of another chemical entity, e.g., a ligand to theconstituent ring.

The iRNA may be conjugated to a ligand via a carrier, wherein thecarrier can be cyclic group or acyclic group. In some embodiments, thecyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, anddecalin. In some embodiments, the acyclic group is a serinol backbone ordiethanolamine backbone.

i. Thermally Destabilizing Modifications

In certain embodiments, a dsRNA molecule can be optimized for RNAinterference by incorporating thermally destabilizing modifications inthe seed region of the antisense strand. As used herein “seed region”means at positions 2-9 of the 5′-end of the referenced strand. Forexample, thermally destabilizing modifications can be incorporated inthe seed region of the antisense strand to reduce or inhibit off-targetgene silencing.

The term “thermally destabilizing modification(s)” includesmodification(s) that would result with a dsRNA with a lower overallmelting temperature (Tm) than the Tm of the dsRNA without having suchmodification(s). For example, the thermally destabilizingmodification(s) can decrease the Tm of the dsRNA by 1-4° C., such asone, two, three or four degrees Celcius. And, the term “thermallydestabilizing nucleotide” refers to a nucleotide containing one or morethermally destabilizing modifications.

It has been discovered that dsRNAs with an antisense strand comprisingat least one thermally destabilizing modification of the duplex withinthe first 9 nucleotide positions, counting from the 5′ end, of theantisense strand have reduced off-target gene silencing activity.Accordingly, in some embodiments, the antisense strand comprises atleast one (e.g., one, two, three, four, five or more) thermallydestabilizing modification of the duplex within the first 9 nucleotidepositions of the 5′ region of the antisense strand. In some embodiments,one or more thermally destabilizing modification(s) of the duplex is/arelocated in positions 2-9, such as, positions 4-8, from the 5′-end of theantisense strand. In some further embodiments, the thermallydestabilizing modification(s) of the duplex is/are located at position6, 7 or 8 from the 5′-end of the antisense strand. In still some furtherembodiments, the thermally destabilizing modification of the duplex islocated at position 7 from the 5′-end of the antisense strand. In someembodiments, the thermally destabilizing modification of the duplex islocated at position 2, 3, 4, 5 or 9 from the 5′-end of the antisensestrand.

An iRNA agent comprises a sense strand and an antisense strand, eachstrand having 14 to 40 nucleotides. The RNAi agent may be represented byformula (L):

In formula (L), B1, B2, B3, B1′, B2′, B3′, and B4′ each areindependently a nucleotide containing a modification selected from thegroup consisting of 2′-O-alkyl, 2′-substituted alkoxy, 2′-substitutedalkyl, 2′-halo, ENA, and BNA/LNA. In one embodiment, B1, B2, B3, B1′,B2′, B3′, and B4′ each contain 2′-OMe modifications. In one embodiment,B1, B2, B3, B1′, B2′, B3′, and B4′ each contain 2′-OMe or 2′-Fmodifications. In one embodiment, at least one of B1, B2, B3, B1′, B2′,B3′, and B4′ contain 2′-O—N-methylacetamido (2′-O-NMA,2′O—CH2C(O)N(Me)H) modification.

C1 is a thermally destabilizing nucleotide placed at a site opposite tothe seed region of the antisense strand (i.e., at positions 2-8 of the5′-end of the antisense strand). For example, C1 is at a position of thesense strand that pairs with a nucleotide at positions 2-8 of the 5′-endof the antisense strand. In one example, C1 is at position 15 from the5′-end of the sense strand. C1 nucleotide bears the thermallydestabilizing modification which can include abasic modification;mismatch with the opposing nucleotide in the duplex; and sugarmodification such as 2′-deoxy modification or acyclic nucleotide e.g.,unlocked nucleic acids (UNA) or glycerol nucleic acid (GNA). In oneembodiment, C1 has thermally destabilizing modification selected fromthe group consisting of: i) mismatch with the opposing nucleotide in theantisense strand; ii) abasic modification selected from the groupconsisting of:

and iii) sugar modification selected from the group consisting of:

wherein B is a modified or unmodified nucleobase, R¹ and R²independently are H, halogen, OR₃, or alkyl; and R₃ is H, alkyl,cycloalkyl, aryl, aralkyl, heteroaryl or sugar. In one embodiment, thethermally destabilizing modification in C1 is a mismatch selected fromthe group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T,U:U, T:T, and U:T; and optionally, at least one nucleobase in themismatch pair is a 2′-deoxy nucleobase. In one example, the thermallydestabilizing modification in C1 is GNA or

T1, T1′, T2′, and T3′ each independently represent a nucleotidecomprising a modification providing the nucleotide a steric bulk that isless or equal to the steric bulk of a 2′-OMe modification. A steric bulkrefers to the sum of steric effects of a modification. Methods fordetermining steric effects of a modification of a nucleotide are knownto one skilled in the art. The modification can be at the 2′ position ofa ribose sugar of the nucleotide, or a modification to a non-ribosenucleotide, acyclic nucleotide, or the backbone of the nucleotide thatis similar or equivalent to the 2′ position of the ribose sugar, andprovides the nucleotide a steric bulk that is less than or equal to thesteric bulk of a 2′-OMe modification. For example, T1, T1′, T2′, and T3′are each independently selected from DNA, RNA, LNA, 2′-F, and2′-F-5′-methyl. In one embodiment, T1 is DNA. In one embodiment, T1′ isDNA, RNA or LNA. In one embodiment, T2′ is DNA or RNA. In oneembodiment, T3′ is DNA or RNA.n¹, n³, and q¹ are independently 4 to 15 nucleotides in length.n⁵, q³, and q⁷ are independently 1-6 nucleotide(s) in length.n⁴, q², and q⁶ are independently 1-3 nucleotide(s) in length;alternatively, n⁴ is 0.q⁵ is independently 0-10 nucleotide(s) in length.n² and q⁴ are independently 0-3 nucleotide(s) in length.

Alternatively, n⁴ is 0-3 nucleotide(s) in length.

In one embodiment, n⁴ can be 0. In one example, n⁴ is 0, and q² and q⁶are 1. In another example, n⁴ is 0, and q² and q⁶ are 1, with twophosphorothioate internucleotide linkage modifications within position1-5 of the sense strand (counting from the 5′-end of the sense strand),and two phosphorothioate internucleotide linkage modifications atpositions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end of the antisense strand).

In one embodiment, n⁴, q², and q⁶ are each 1.

In one embodiment, n², n⁴, q², q⁴, and q⁶ are each 1.

In one embodiment, C1 is at position 14-17 of the 5′-end of the sensestrand, when the sense strand is 19-22 nucleotides in length, and n⁴is 1. In one embodiment, C1 is at position 15 of the 5′-end of the sensestrand

In one embodiment, T3′ starts at position 2 from the 5′ end of theantisense strand. In one example, T3′ is at position 2 from the 5′ endof the antisense strand and q⁶ is equal to 1.

In one embodiment, T1′ starts at position 14 from the 5′ end of theantisense strand. In one example, T1′ is at position 14 from the 5′ endof the antisense strand and q² is equal to 1.

In an exemplary embodiment, T3′ starts from position 2 from the 5′ endof the antisense strand and T1′ starts from position 14 from the 5′ endof the antisense strand. In one example, T3′ starts from position 2 fromthe 5′ end of the antisense strand and q⁶ is equal to 1 and T1′ startsfrom position 14 from the 5′ end of the antisense strand and q² is equalto 1.

In one embodiment, T1′ and T3′ are separated by 11 nucleotides in length(i.e. not counting the T1′ and T3′ nucleotides).

In one embodiment, T1′ is at position 14 from the 5′ end of theantisense strand. In one example, T1′ is at position 14 from the 5′ endof the antisense strand and q² is equal to 1, and the modification atthe 2′ position or positions in a non-ribose, acyclic or backbone thatprovide less steric bulk than a 2′-OMe ribose.

In one embodiment, T3′ is at position 2 from the 5′ end of the antisensestrand. In one example, T3′ is at position 2 from the 5′ end of theantisense strand and q⁶ is equal to 1, and the modification at the 2′position or positions in a non-ribose, acyclic or backbone that provideless than or equal to steric bulk than a 2′-OMe ribose.

In one embodiment, T1 is at the cleavage site of the sense strand. Inone example, T1 is at position 11 from the 5′ end of the sense strand,when the sense strand is 19-22 nucleotides in length, and n² is 1. In anexemplary embodiment, T1 is at the cleavage site of the sense strand atposition 11 from the 5′ end of the sense strand, when the sense strandis 19-22 nucleotides in length, and n² is 1, In one embodiment, T2′starts at position 6 from the 5′ end of the antisense strand. In oneexample, T2′ is at positions 6-10 from the 5′ end of the antisensestrand, and q⁴ is 1.

In an exemplary embodiment, T1 is at the cleavage site of the sensestrand, for instance, at position 11 from the 5′ end of the sensestrand, when the sense strand is 19-22 nucleotides in length, and n² is1; T1′ is at position 14 from the 5′ end of the antisense strand, and q²is equal to 1, and the modification to T1′ is at the 2′ position of aribose sugar or at positions in a non-ribose, acyclic or backbone thatprovide less steric bulk than a 2′-OMe ribose; T2′ is at positions 6-10from the 5′ end of the antisense strand, and q⁴ is 1; and T3′ is atposition 2 from the 5′ end of the antisense strand, and q⁶ is equal to1, and the modification to T3′ is at the 2′ position or at positions ina non-ribose, acyclic or backbone that provide less than or equal tosteric bulk than a 2′-OMe ribose.

In one embodiment, T2′ starts at position 8 from the 5′ end of theantisense strand. In one example, T2′ starts at position 8 from the 5′end of the antisense strand, and q⁴ is 2.

In one embodiment, T2′ starts at position 9 from the 5′ end of theantisense strand. In one example, T2′ is at position 9 from the 5′ endof the antisense strand, and q⁴ is 1.

In one embodiment, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1,B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 1, B3′ is 2′-OMe or2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁴ is 1; withtwo phosphorothioate internucleotide linkage modifications withinpositions 1-5 of the sense strand (counting from the 5′-end of the sensestrand), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end of the antisense strand).

In one embodiment, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q⁴ is 4, T2′is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within positions 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n⁵ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n¹ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within positions 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 6, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 7, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 6, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 7, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q⁴ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within positions 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q⁴ is 4, T2′is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within positions 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 5, T2′is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1; optionally with at least 2 additional T atthe 3′-end of the antisense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 5, T2′is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1; optionally with at least 2 additional T atthe 3′-end of the antisense strand; with two phosphorothioateinternucleotide linkage modifications within positions 1-5 of the sensestrand (counting from the 5′-end of the sense strand), and twophosphorothioate internucleotide linkage modifications at positions 1and 2 and two phosphorothioate internucleotide linkage modificationswithin positions 18-23 of the antisense strand (counting from the 5′-endof the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q⁴ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within positions 1-5 of the sense strand (counting fromthe 5′-end), and two phosphorothioate internucleotide linkagemodifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁴ is 1. In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁴ is 1; with twophosphorothioate internucleotide linkage modifications within positions1-5 of the sense strand (counting from the 5′-end of the sense strand),and two phosphorothioate internucleotide linkage modifications atpositions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end of the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁴ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q⁴ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within positions 1-5 of the sense strand (counting fromthe 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand).

The RNAi agent can comprise a phosphorus-containing group at the 5′-endof the sense strand or antisense strand. The 5′-endphosphorus-containing group can be 5′-end phosphate (5′-P), 5′-endphosphorothioate (5′-PS), 5′-end phosphorodithioate (5′-PS₂), 5′-endvinylphosphonate (5′-VP), 5′-end methylphosphonate (MePhos), or5′-deoxy-5′-C-malonyl

When the 5′-end phosphorus-containing group is 5′-end vinylphosphonate(5′-VP), the 5′-VP can be either 5′-E-VP isomer (i.e.,trans-vinylphosphonate,

5′-Z-VP isomer (i.e., cis-vinylphosphonate,

or mixtures thereof.In one embodiment, the RNAi agent comprises a phosphorus-containinggroup at the 5′-end of the sense strand. In one embodiment, the RNAiagent comprises a phosphorus-containing group at the 5′-end of theantisense strand.

In one embodiment, the RNAi agent comprises a 5′-P. In one embodiment,the RNAi agent comprises a 5′-P in the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-PS. In one embodiment,the RNAi agent comprises a 5′-PS in the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-VP. In one embodiment,the RNAi agent comprises a 5′-VP in the antisense strand. In oneembodiment, the RNAi agent comprises a 5′-E-VP in the antisense strand.In one embodiment, the RNAi agent comprises a 5′-Z-VP in the antisensestrand.

In one embodiment, the RNAi agent comprises a 5′-PS₂. In one embodiment,the RNAi agent comprises a 5′-PS₂ in the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-PS₂. In one embodiment,the RNAi agent comprises a 5′-deoxy-5′-C-malonyl in the antisensestrand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1. The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1. The RNAi agent also comprises a 5′-VP. The5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1. The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1. The RNAi agent also comprises a5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-VP. The 5′-VP may be5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n³ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁴ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1. The dsRNA agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1. The RNAi agent also comprises a 5′-VP. The 5′-VPmay be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1. The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1. The RNAi agent also comprises a5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-VP. The 5′-VP maybe 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁴ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁴ is 1. The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁴ is 1. The RNAi agent also comprises a 5′-VP. The5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁴ is 1. The dsRNAi RNA agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁴ is 1. The RNAi agent also comprises a5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n³ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-VP. The 5′-VP may be5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁴ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁴ is 1. The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁴ is 1. The RNAi agent also comprises a 5′-VP. The 5′-VP may be5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁴ is 1. The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁴ is 1. The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n³ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁴ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, orcombination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-P and a targeting ligand. Inone embodiment, the 5′-P is at the 5′-end of the antisense strand, andthe targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS and a targeting ligand.In one embodiment, the 5′-PS is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP,5′-Z-VP, or combination thereof), and a targeting ligand.

In one embodiment, the 5′-VP is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS₂ and a targeting ligand.In one embodiment, the 5′-PS₂ is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and atargeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the5′-end of the antisense strand, and the targeting ligand is at the3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-P and a targetingligand. In one embodiment, the 5′-P is at the 5′-end of the antisensestrand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-PS and a targetingligand. In one embodiment, the 5′-PS is at the 5′-end of the antisensestrand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-VP (e.g., a5′-E-VP, 5′-Z-VP, or combination thereof) and a targeting ligand. In oneembodiment, the 5′-VP is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-PS₂ and a targetingligand. In one embodiment, the 5′-PS₂ is at the 5′-end of the antisensestrand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is2′-OMe, and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyland a targeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl isat the 5′-end of the antisense strand, and the targeting ligand is atthe 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n³ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-P and a targeting ligand. Inone embodiment, the 5′-P is at the 5′-end of the antisense strand, andthe targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS and a targeting ligand.In one embodiment, the 5′-PS is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP,5′-Z-VP, or combination thereof) and a targeting ligand. In oneembodiment, the 5′-VP is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁴ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-PS₂ and a targeting ligand.In one embodiment, the 5′-PS₂ is at the 5′-end of the antisense strand,and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1,B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotidelinkage modifications within position 1-5 of the sense strand (countingfrom the 5′-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and twophosphorothioate internucleotide linkage modifications within positions18-23 of the antisense strand (counting from the 5′-end of the antisensestrand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and atargeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the5′-end of the antisense strand, and the targeting ligand is at the3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-P and a targeting ligand. In oneembodiment, the 5′-P is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-PS and a targeting ligand. In oneembodiment, the 5′-PS is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP, 5′-Z-VP, orcombination thereof) and a targeting ligand. In one embodiment, the5′-VP is at the 5′-end of the antisense strand, and the targeting ligandis at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁷ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-PS₂ and a targeting ligand. In oneembodiment, the 5′-PS₂ is at the 5′-end of the antisense strand, and thetargeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F,and q⁴ is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the5′-end of the sense strand), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end of the antisense strand). TheRNAi agent also comprises a 5′-deoxy-5′-C-malonyl and a targetingligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the 5′-end ofthe antisense strand, and the targeting ligand is at the 3′-end of thesense strand.

In a particular embodiment, an RNAi agent of the present inventioncomprises:

-   -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker; and        -   (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11,            13, 17, 19, and 21, and 2′-OMe modifications at positions 2,            4, 6, 8, 12, 14 to 16, 18, and 20 (counting from the 5′            end); and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii)2′-OMe modifications at positions 1, 3, 5, 9, 11 to 13,            15, 17, 19, 21, and 23, and 2′F modifications at positions            2, 4, 6 to 8, 10, 14, 16, 18, 20, and 22 (counting from the            5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 21 and 22, and between nucleotide            positions 22 and 23 (counting from the 5′ end);    -   wherein the dsRNA agents have a two nucleotide overhang at the        3′-end of the antisense strand, and a blunt end at the 5′-end of        the antisense strand.

In another particular embodiment, an RNAi agent of the present inventioncomprises:

-   -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11,            13, 15, 17, 19, and 21, and 2′-OMe modifications at            positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from            the 5′ end); and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end);    -   and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii)2′-OMe modifications at positions 1, 3, 5, 7, 9, 11 to            13, 15, 17, 19, and 21 to 23, and 2′F modifications at            positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from            the 5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present inventioncomprises:

-   -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-OMe modifications at positions 1 to 6, 8, 10, and            12 to 21, 2′-F modifications at positions 7, and 9, and a            deoxy-nucleotide (e.g. dT) at position 11 (counting from the            5′ end); and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end);    -   and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii)2′-OMe modifications at positions 1, 3, 7, 9, 11, 13,            15, 17, and 19 to 23, and 2′-F modifications at positions 2,            4 to 6, 8, 10, 12, 14, 16, and 18 (counting from the 5′            end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present inventioncomprises:

-   -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-OMe modifications at positions 1 to 6, 8, 10, 12,            14, and 16 to 21, and 2′-F modifications at positions 7, 9,            11, 13, and 15; and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end);    -   and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii)2′-OMe modifications at positions 1, 5, 7, 9, 11, 13,            15, 17, 19, and 21 to 23, and 2′-F modifications at            positions 2 to 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting            from the 5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present inventioncomprises:

-   -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-OMe modifications at positions 1 to 9, and 12 to            21, and 2′-F modifications at positions 10, and 11; and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end);    -   and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii)2′-OMe modifications at positions 1, 3, 5, 7, 9, 11 to            13, 15, 17, 19, and 21 to 23, and 2′-F modifications at            positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from            the 5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present inventioncomprises:

-   -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11,            and 13, and 2′-OMe modifications at positions 2, 4, 6, 8,            12, and 14 to 21; and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end);    -   and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii)2′-OMe modifications at positions 1, 3, 5 to 7, 9, 11 to            13, 15, 17 to 19, and 21 to 23, and 2′-F modifications at            positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5′            end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present inventioncomprises:

-   -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-OMe modifications at positions 1, 2, 4, 6, 8, 12,            14, 15, 17, and 19 to 21, and 2′-F modifications at            positions 3, 5, 7, 9 to 11, 13, 16, and 18; and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end);    -   and    -   (b) an antisense strand having:        -   (i) a length of 25 nucleotides;        -   (ii)2′-OMe modifications at positions 1, 4, 6, 7, 9, 11 to            13, 15, 17, and 19 to 23, 2′-F modifications at positions 2,            3, 5, 8, 10, 14, 16, and 18, and desoxy-nucleotides (e.g.            dT) at positions 24 and 25 (counting from the 5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a four nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present inventioncomprises:

-   -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-OMe modifications at positions 1 to 6, 8, and 12 to            21, and 2′-F modifications at positions 7, and 9 to 11; and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end);    -   and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii)2′-OMe modifications at positions 1, 3 to 5, 7, 8, 10 to            13, 15, and 17 to 23, and 2′-F modifications at positions 2,            6, 9, 14, and 16 (counting from the 5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present inventioncomprises:

-   -   (a) a sense strand having:        -   (i) a length of 21 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-OMe modifications at positions 1 to 6, 8, and 12 to            21, and 2′-F modifications at positions 7, and 9 to 11; and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end);    -   and    -   (b) an antisense strand having:        -   (i) a length of 23 nucleotides;        -   (ii)2′-OMe modifications at positions 1, 3 to 5, 7, 10 to            13, 15, and 17 to 23, and 2′-F modifications at positions 2,            6, 8, 9, 14, and 16 (counting from the 5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 21 and 22, and between            nucleotide positions 22 and 23 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present inventioncomprises:

-   -   (a) a sense strand having:        -   (i) a length of 19 nucleotides;        -   (ii) an ASGPR ligand attached to the 3′-end, wherein said            ASGPR ligand comprises three GalNAc derivatives attached            through a trivalent branched linker;        -   (iii) 2′-OMe modifications at positions 1 to 4, 6, and 10 to            19, and 2′-F modifications at positions 5, and 7 to 9; and        -   (iv) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, and between nucleotide            positions 2 and 3 (counting from the 5′ end);    -   and    -   (b) an antisense strand having:        -   (i) a length of 21 nucleotides;        -   (ii)2′-OMe modifications at positions 1, 3 to 5, 7, 10 to            13, 15, and 17 to 21, and 2′-F modifications at positions 2,            6, 8, 9, 14, and 16 (counting from the 5′ end); and        -   (iii) phosphorothioate internucleotide linkages between            nucleotide positions 1 and 2, between nucleotide positions 2            and 3, between nucleotide positions 19 and 20, and between            nucleotide positions 20 and 21 (counting from the 5′ end);            wherein the RNAi agents have a two nucleotide overhang at            the 3′-end of the antisense strand, and a blunt end at the            5′-end of the antisense strand.

In certain embodiments, the iRNA for use in the methods of the inventionis an agent selected from agents listed in any one of Tables 2-3 and6-7. These agents may further comprise a ligand.

III. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involveschemically linking to the iRNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution, or cellularuptake of the iRNA e.g., into a cell. Such moieties include but are notlimited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556). In otherembodiments, the ligand is cholic acid (Manoharan et al., Biorg. Med.Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan etal., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990,259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In certain embodiments, a ligand alters the distribution, targeting, orlifetime of an iRNA agent into which it is incorporated. In someembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. In some embodiments, ligandsdo not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, orhyaluronic acid); or a lipid. The ligand can also be a recombinant orsynthetic molecule, such as a synthetic polymer, e.g., a syntheticpolyamino acid. Examples of polyamino acids include polyamino acid is apolylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic. In certain embodiments, the ligand is amultivalent galactose, e.g., an N-acetyl-galactosamine.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, or intermediate filaments. The drug can be, for example,taxol, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins, etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.,oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases,comprising multiple of phosphorothioate linkages in the backbone arealso amenable to the present invention as ligands (e.g. as PK modulatingligands). In addition, aptamers that bind serum components (e.g. serumproteins) are also suitable for use as PK modulating ligands in theembodiments described herein.

Ligand-conjugated iRNAs of the invention may be synthesized by the useof an oligonucleotide that bears a pendant reactive functionality, suchas that derived from the attachment of a linking molecule onto theoligonucleotide (described below). This reactive oligonucleotide may bereacted directly with commercially-available ligands, ligands that aresynthesized bearing any of a variety of protecting groups, or ligandsthat have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems® (Foster City,Calif.). Any other methods for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated iRNAs and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conjugates

In certain embodiments, the ligand or conjugate is a lipid orlipid-based molecule. In some embodiments, such a lipid or lipid-basedmolecule binds a serum protein, e.g., human serum albumin (HSA). An HSAbinding ligand allows for distribution of the conjugate to a targettissue, e.g., a non-kidney target tissue of the body. For example, thetarget tissue can be the liver, including parenchymal cells of theliver. Other molecules that can bind HSA can also be used as ligands.For example, naproxen or aspirin can be used. A lipid or lipid-basedligand can (a) increase resistance to degradation of the conjugate, (b)increase targeting or transport into a target cell or cell membrane, or(c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In certain embodiments, the lipid based ligand binds HSA. In someembodiments, it binds HSA with a sufficient affinity such that theconjugate will be distributed to a non-kidney tissue. However, it ispreferred that the affinity not be so strong that the HSA-ligand bindingcannot be reversed.

In other embodiments, the lipid based ligand binds HSA weakly or not atall, such that the conjugate will be distributed to the kidney. Othermoieties that target to kidney cells can also be used in place of, or inaddition to, the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells.

Exemplary vitamins include vitamin A, E, and K. Other exemplary vitaminsinclude are B vitamin, e.g., folic acid, B12, riboflavin, biotin,pyridoxal or other vitamins or nutrients taken up by target cells suchas liver cells. Also included are HSA and low density lipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, such as, ahelical cell-permeation agent. In some embodiments, the agent isamphipathic. An exemplary agent is a peptide such as tat orantennopedia. If the agent is a peptide, it can be modified, including apeptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages,and use of D-amino acids. In some embodiments, the helical agent is analpha-helical agent, which has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 17). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 18) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 19) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 20)have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glycosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand, e.g., PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, aniRNA further comprises a carbohydrate. The carbohydrate conjugated iRNAis advantageous for the in vivo delivery of nucleic acids, as well ascompositions suitable for in vivo therapeutic use, as described herein.As used herein, “carbohydrate” refers to a compound which is either acarbohydrate per se made up of one or more monosaccharide units havingat least 6 carbon atoms (which can be linear, branched or cyclic) withan oxygen, nitrogen or sulfur atom bonded to each carbon atom; or acompound having as a part thereof a carbohydrate moiety made up of oneor more monosaccharide units each having at least six carbon atoms(which can be linear, branched or cyclic), with an oxygen, nitrogen orsulfur atom bonded to each carbon atom. Representative carbohydratesinclude the sugars (mono-, di-, tri-, and oligosaccharides containingfrom about 4, 5, 6, 7, 8, or 9 monosaccharide units), andpolysaccharides such as starches, glycogen, cellulose and polysaccharidegums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7,or C8) sugars; di- and trisaccharides include sugars having two or threemonosaccharide units (e.g., C5, C6, C7, or C8).

In certain embodiments, a carbohydrate conjugate for use in thecompositions and methods of the invention is a monosaccharide.

In certain embodiments, the monosaccharide is an N-acetylgalactosamine(GalNAc). GalNAc conjugates, which comprise one or moreN-acetylgalactosamine (GalNAc) derivatives, are described, for example,in U.S. Pat. No. 8,106,022, the entire content of which is herebyincorporated herein by reference. In some embodiments, the GalNAcconjugate serves as a ligand that targets the iRNA to particular cells.In some embodiments, the GalNAc conjugate targets the iRNA to livercells, e.g., by serving as a ligand for the asialoglycoprotein receptorof liver cells (e.g., hepatocytes).

In some embodiments, the carbohydrate conjugate comprises one or moreGalNAc derivatives. The GalNAc derivatives may be attached via a linker,e.g., a bivalent or trivalent branched linker. In some embodiments theGalNAc conjugate is conjugated to the 3′ end of the sense strand. Insome embodiments, the GalNAc conjugate is conjugated to the iRNA agent(e.g., to the 3′ end of the sense strand) via a linker, e.g., a linkeras described herein. In some embodiments the GalNAc conjugate isconjugated to the 5′ end of the sense strand. In some embodiments, theGalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5′ end ofthe sense strand) via a linker, e.g., a linker as described herein.

In certain embodiments of the invention, the GalNAc or GalNAc derivativeis attached to an iRNA agent of the invention via a monovalent linker.In some embodiments, the GalNAc or GalNAc derivative is attached to aniRNA agent of the invention via a bivalent linker. In yet otherembodiments of the invention, the GalNAc or GalNAc derivative isattached to an iRNA agent of the invention via a trivalent linker. Inother embodiments of the invention, the GalNAc or GalNAc derivative isattached to an iRNA agent of the invention via a tetravalent linker.

In certain embodiments, the double stranded RNAi agents of the inventioncomprise one GalNAc or GalNAc derivative attached to the iRNA agent. Incertain embodiments, the double stranded RNAi agents of the inventioncomprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAcderivatives, each independently attached to a plurality of nucleotidesof the double stranded RNAi agent through a plurality of monovalentlinkers.

In some embodiments, for example, when the two strands of an iRNA agentof the invention are part of one larger molecule connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′-end of the respective other strand forming a hairpin loopcomprising, a plurality of unpaired nucleotides, each unpairednucleotide within the hairpin loop may independently comprise a GalNAcor GalNAc derivative attached via a monovalent linker. The hairpin loopmay also be formed by an extended overhang in one strand of the duplex.

In some embodiments, for example, when the two strands of an iRNA agentof the invention are part of one larger molecule connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′-end of the respective other strand forming a hairpin loopcomprising, a plurality of unpaired nucleotides, each unpairednucleotide within the hairpin loop may independently comprise a GalNAcor GalNAc derivative attached via a monovalent linker. The hairpin loopmay also be formed by an extended overhang in one strand of the duplex.

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is selected from the group consisting of:

wherein Y is O or S and n is 3-6 (Formula XXIV);

wherein Y is O or S and n is 3-6 (Formula XXV);

wherein X is O or S

In another embodiment, a carbohydrate conjugate for use in thecompositions and methods of the invention is a monosaccharide. In oneembodiment, the monosaccharide is an N-acetylgalactosamine, such as

In some embodiments, the RNAi agent is attached to the carbohydrateconjugate via a linker as shown in the following schematic, wherein X isO or S

In some embodiments, the RNAi agent is conjugated to L96 as defined inTable 1 and shown below:

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

(Formula XXXVI), when one of X or Y is an oligonucleotide, the other isa hydrogen.

In some embodiments, a suitable ligand is a ligand disclosed in WO2019/055633, the entire contents of which are incorporated herein byreference. In one embodiment the ligand comprises the structure below:

In certain embodiments of the invention, the GalNAc or GalNAc derivativeis attached to an iRNA agent of the invention via a monovalent linker.In some embodiments, the GalNAc or GalNAc derivative is attached to aniRNA agent of the invention via a bivalent linker. In yet otherembodiments of the invention, the GalNAc or GalNAc derivative isattached to an iRNA agent of the invention via a trivalent linker.

In one embodiment, the double stranded RNAi agents of the inventioncomprise one or more GalNAc or GalNAc derivative attached to the iRNAagent. The GalNAc may be attached to any nucleotide via a linker on thesense strand or antsisense strand. The GalNac may be attached to the5′-end of the sense strand, the 3′ end of the sense strand, the 5′-endof the antisense strand, or the 3′-end of the antisense strand. In oneembodiment, the GalNAc is attached to the 3′ end of the sense strand,e.g., via a trivalent linker.

In other embodiments, the double stranded RNAi agents of the inventioncomprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAcderivatives, each independently attached to a plurality of nucleotidesof the double stranded RNAi agent through a plurality of linkers, e.g.,monovalent linkers.

In some embodiments, for example, when the two strands of an iRNA agentof the invention is part of one larger molecule connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′-end of the respective other strand forming a hairpin loopcomprising, a plurality of unpaired nucleotides, each unpairednucleotide within the hairpin loop may independently comprise a GalNAcor GalNAc derivative attached via a monovalent linker.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator or a cell permeation peptide.

Additional carbohydrate conjugates and linkers suitable for use in thepresent invention include those described in PCT Publication Nos. WO2014/179620 and WO 2014/179627, the entire contents of each of which areincorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an iRNA oligonucleotide with various linkers that can becleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, or substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic, orsubstituted aliphatic. In one embodiment, the linker is about 1-24atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16,7-17, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In one embodiment, thecleavable linking group is cleaved at least about 10 times, 20, times,30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, ormore, or at least 100 times faster in a target cell or under a firstreference condition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential, or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a selected pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In some embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100times faster in the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood or serum (or under invitro conditions selected to mimic extracellular conditions).

i. Redox Cleavable Linking Groups

In certain embodiments, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular iRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In other embodiments, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—, wherein Rk at eachoccurrence can be, independently, C1-C20 alkyl, C1-C20 haloalkyl, C6-C10aryl, or C7-C12 aralkyl. Exemplary embodiments include are—O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—,—S—P(O)(H)—S—, and —O—P(S)(H)—S—. In certain embodiments, aphosphate-based linking group is —O—P(O)(OH)—O—. These candidates can beevaluated using methods analogous to those described above.

iii. Acid Cleavable Linking Groups

In other embodiments, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In some embodiments acid cleavablelinking groups are cleaved in an acidic environment with a pH of about6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such asenzymes that can act as a general acid. In a cell, specific low pHorganelles, such as endosomes and lysosomes can provide a cleavingenvironment for acid cleavable linking groups. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C≡NN—, C(O)O, or —OC(O). An exemplary embodiment iswhen the carbon attached to the oxygen of the ester (the alkoxy group)is an aryl group, substituted alkyl group, or tertiary alkyl group suchas dimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In other embodiments, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include, but are not limited to,esters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet other embodiments, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In some embodiments, an iRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of iRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include, but are not limited to,

(Formula XLIV), when one of X or Y is an oligonucleotide, the other is ahydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more “GaINAc” (N-acetylgalactosamnine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalentor trivalent branched linker selected from the group of structures shownin any of formula (XLV)-(XLVIII):

wherein:q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5 q5C represent independently foreach occurrence 0-20 and wherein the repeating unit can be the same ordifferent;P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl; L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A),L^(5B) and L^(5C) represent the ligand; i.e. each independently for eachoccurrence a monosaccharide (such as GalNAc), disaccharide,trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; andR^(a) is H or amino acid side chain. Trivalent conjugating GalNAcderivatives are particularly useful for use with RNAi agents forinhibiting the expression of a target gene, such as those of formula(XLIX):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such asGalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. Patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; and 8,106,022, the entire contents of each ofwhich are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of thisinvention, are iRNA compounds, such as, dsRNAi agents, that contain twoor more chemically distinct regions, each made up of at least onemonomer unit, i.e., a nucleotide in the case of a dsRNA compound. TheseiRNAs typically contain at least one region wherein the RNA is modifiedso as to confer upon the iRNA increased resistance to nucleasedegradation, increased cellular uptake, or increased binding affinityfor the target nucleic acid. An additional region of the iRNA can serveas a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of iRNA inhibition of gene expression.Consequently, comparable results can often be obtained with shorteriRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA targetcan be routinely detected by gel electrophoresis and, if necessary,associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof RNAs bearing an aminolinker at one or more positions of the sequence.The amino group is then reacted with the molecule being conjugated usingappropriate coupling or activating reagents. The conjugation reactioncan be performed either with the RNA still bound to the solid support orfollowing cleavage of the RNA, in solution phase. Purification of theRNA conjugate by HPLC typically affords the pure conjugate.

IV. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within asubject, such as a human subject (e.g., a subject in need thereof, suchas a subject susceptible to or diagnosed with a xanthinedehydrogenase-associated disorder) can be achieved in a number ofdifferent ways. For example, delivery may be performed by contacting acell with an iRNA of the invention either in vitro or in vivo. In vivodelivery may also be performed directly by administering a compositioncomprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivodelivery may be performed indirectly by administering one or morevectors that encode and direct the expression of the iRNA. Thesealternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. RNAinterference has also shown success with local delivery to the CNS bydirect injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMCNeurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528;Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602).Modification of the RNA or the pharmaceutical carrier can also permittargeting of the iRNA to the target tissue and avoid undesirableoff-target effects. iRNA molecules can be modified by chemicalconjugation to lipophilic groups such as cholesterol to enhance cellularuptake and prevent degradation. For example, an iRNA directed againstApoB conjugated to a lipophilic cholesterol moiety was injectedsystemically into mice and resulted in knockdown of apoB mRNA in boththe liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178).

In an alternative embodiment, the iRNA can be delivered using drugdelivery systems such as a nanoparticle, a dendrimer, a polymer,liposomes, or a cationic delivery system. Positively charged cationicdelivery systems facilitate binding of an iRNA molecule (negativelycharged) and also enhance interactions at the negatively charged cellmembrane to permit efficient uptake of an iRNA by the cell. Cationiclipids, dendrimers, or polymers can either be bound to an iRNA, orinduced to form a vesicle or micelle (see e.g., Kim S H, et al (2008)Journal of Controlled Release 129(2):107-116) that encases an iRNA. Theformation of vesicles or micelles further prevents degradation of theiRNA when administered systemically. Methods for making andadministering cationic-iRNA complexes are well within the abilities ofone skilled in the art (see e.g., Sorensen, D R, et al (2003) J. Mol.Biol 327:761-766; Verma, U N, et al (2003) Clin. Cancer Res.9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, whichare incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N, et al (2003), supra), “solid nucleic acid lipid particles”(Zimmermann, T S, et al (2006) Nature 441:111-114), cardiolipin (Chien,P Y, et al (2005) Cancer Gene Ther. 12:321-328; Pal, A, et al (2005) IntJ. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E, et al (2008)Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed.Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol.Pharm. 3:472-487), and polyamidoamines (Tomalia, D A, et al (2007)Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res.16:1799-1804). In some embodiments, an iRNA forms a complex withcyclodextrin for systemic administration. Methods for administration andpharmaceutical compositions of iRNAs and cyclodextrins can be found inU.S. Pat. No. 7,427,605, which is herein incorporated by reference inits entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the xanthine dehydrogenase gene can be expressed fromtranscription units inserted into DNA or RNA vectors (see, e.g.,Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A, et al.,International PCT Publication No. WO 00/22113, Conrad, International PCTPublication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299).Expression can be transient (on the order of hours to weeks) orsustained (weeks to months or longer), depending upon the specificconstruct used and the target tissue or cell type. These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be an integrating or non-integrating vector. Thetransgene can also be constructed to permit it to be inherited as anextrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA(1995) 92:1292).

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are known in the art.

V. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful forpreventing or treating a xanthine dehydrogenase-associated disorder.Such pharmaceutical compositions are formulated based on the mode ofdelivery. One example is compositions that are formulated for systemicadministration via parenteral delivery, e.g., by subcutaneous (SC),intramuscular (IM), or intravenous (IV) delivery. The pharmaceuticalcompositions of the invention may be administered in dosages sufficientto inhibit expression of a xanthine dehydrogenase gene.

In some embodiments, the pharmaceutical compositions of the inventionare sterile. In another embodiment, the pharmaceutical compositions ofthe invention are pyrogen free.

The pharmaceutical compositions of the invention may be administered indosages sufficient to inhibit expression of a xanthine dehydrogenasegene. In general, a suitable dose of an iRNA of the invention will be inthe range of about 0.001 to about 200.0 milligrams per kilogram bodyweight of the recipient per day, generally in the range of about 1 to 50mg per kilogram body weight per day. Typically, a suitable dose of aniRNA of the invention will be in the range of about 0.1 mg/kg to about5.0 mg/kg, or about 0.3 mg/kg to about 3.0 mg/kg. A repeat-dose regimenmay include administration of a therapeutic amount of iRNA on a regularbasis, such as every month, once every 3-6 months, or once a year. Incertain embodiments, the iRNA is administered about once per month toabout once per six months.

After an initial treatment regimen, the treatments can be administeredon a less frequent basis. Duration of treatment can be determined basedon the severity of disease.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that doses are administered at not more than1, 2, 3, or 4 month intervals. In some embodiments of the invention, asingle dose of the pharmaceutical compositions of the invention isadministered about once per month. In other embodiments of theinvention, a single dose of the pharmaceutical compositions of theinvention is administered quarterly (i.e., about every three months). Inother embodiments of the invention, a single dose of the pharmaceuticalcompositions of the invention is administered twice per year (i.e.,about once every six months).

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to mutations present in the subject, previoustreatments, the general health or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a prophylactically ortherapeutically effective amount, as appropriate, of a composition caninclude a single treatment or a series of treatments.

The iRNA can be delivered in a manner to target a particular tissue(e.g., hepatocytes).

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids, and self-emulsifying semisolids. Formulationsinclude those that target the liver.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers.

A. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution either in the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an d/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Other means of stabilizing emulsions entail the use ofemulsifiers that can be incorporated into either phase of the emulsion.Emulsifiers can broadly be classified into four categories: syntheticsurfactants, naturally occurring emulsifiers, absorption bases, andfinely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Formsand Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C.,2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic, and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives, andantioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

The application of emulsion formulations via dermatological, oral, andparenteral routes, and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion canbe defined as a system of water, oil, and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215).

iii. Microparticles

An iRNA of the invention may be incorporated into a particle, e.g., amicroparticle. Microparticles can be produced by spray-drying, but mayalso be produced by other methods including lyophilization, evaporation,fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers and their use in manufacture of pharmaceuticalcompositions and delivery of pharmaceutical agents are well known in theart.

v. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agent,or any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Such agent are well known in the art.

vi. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavorings,or aromatic substances, and the like which do not deleteriously interactwith the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol, or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA and (b) one or more agents whichfunction by a non-iRNA mechanism and which are useful in treating axanthine dehydrogenase-associated disorder.

Toxicity and prophylactic efficacy of such compounds can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose prophylactically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED50, such as, anED80 or ED90, with little or no toxicity. The dosage can vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any compound used in the methods featuredin the invention, the prophylactically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated in animalmodels to achieve a circulating plasma concentration range of thecompound or, when appropriate, of the polypeptide product of a targetsequence (e.g., achieving a decreased concentration of the polypeptide)that includes the IC50 (i.e., the concentration of the test compoundwhich achieves a half-maximal inhibition of symptoms) or higher levelsof inhibition as determined in cell culture. Such information can beused to more accurately determine useful doses in humans. Levels inplasma can be measured, for example, by high performance liquidchromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents used for the prevention or treatment of a xanthinedehydrogenase-associated disorder. In any event, the administeringphysician can adjust the amount and timing of iRNA administration on thebasis of results observed using standard measures of efficacy known inthe art or described herein.

VI. Methods For Inhibiting Xanthine Dehydrogenase Expression

The present invention also provides methods of inhibiting expression ofan XDH gene in a cell. The methods include contacting a cell with anRNAi agent, e.g., double stranded RNA agent, in an amount effective toinhibit expression of XDH in the cell, thereby inhibiting expression ofXDH in the cell.

Contacting of a cell with an iRNA, e.g., a double stranded RNA agent,may be done in vitro or in vivo. Contacting a cell in vivo with the iRNAincludes contacting a cell or group of cells within a subject, e.g., ahuman subject, with the iRNA. Combinations of in vitro and in vivomethods of contacting a cell are also possible. Contacting a cell may bedirect or indirect, as discussed above. Furthermore, contacting a cellmay be accomplished via a targeting ligand, including any liganddescribed herein or known in the art. In some embodiments, the targetingligand is a carbohydrate moiety, e.g., a GalNAc ligand, or any otherligand that directs the RNAi agent to a site of interest.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating”, “suppressing”, and othersimilar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a xanthine dehydrogenase gene” isintended to refer to inhibition of expression of any xanthinedehydrogenase gene (such as, e.g., a mouse xanthine dehydrogenase gene,a rat xanthine dehydrogenase gene, a monkey xanthine dehydrogenase gene,or a human xanthine dehydrogenase gene) as well as variants or mutantsof a xanthine dehydrogenase gene. Thus, the xanthine dehydrogenase genemay be a wild-type xanthine dehydrogenase gene, a mutant xanthinedehydrogenase gene, or a transgenic xanthine dehydrogenase gene in thecontext of a genetically manipulated cell, group of cells, or organism.

“Inhibiting expression of a xanthine dehydrogenase gene” includes anylevel of inhibition of a xanthine dehydrogenase gene, e.g., at leastpartial suppression of the expression of a xanthine dehydrogenase gene,such as, a clinically relevant level of suppression. The expression ofthe xanthine dehydrogenase gene may be assessed based on the level, orthe change in the level, of any variable associated with xanthinedehydrogenase gene expression, e.g., xanthine dehydrogenase mRNA levelor xanthine dehydrogenase protein level, or, for example, serum uricacid levels. Inhibition may be assessed by a decrease in an absolute orrelative level of one or more of these variables compared with a controllevel. This level may be assessed in an individual cell or in a group ofcells, including, for example, a sample derived from a subject. It isunderstood that xanthine dehydrogenase is expressed predominantly in theliver, and is present in circulation.

Inhibition may be assessed by a decrease in an absolute or relativelevel of one or more variables that are associated with xanthinedehydrogenase expression compared with a control level. The controllevel may be any type of control level that is utilized in the art,e.g., a pre-dose baseline level, or a level determined from a similarsubject, cell, or sample that is untreated or treated with a control(such as, e.g., buffer only control or inactive agent control).

In some embodiments of the methods of the invention, expression of axanthine dehydrogenase gene is inhibited by at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95%, or to below the level of detection ofthe assay. In some embodiments, expression of a xanthine dehydrogenasegene is inhibited by at least 70%. It is further understood thatinhibition of xanthine dehydrogenase expression in certain tissues,e.g., in gall bladder, without a significant inhibition of expression inother tissues, e.g., brain, may be desirable. In some embodiments,expression level is determined using the assay method provided inExample 2 with a 10 nM siRNA concentration in the appropriate speciesmatched cell line.

In certain embodiments, inhibition of expression in vivo is determinedby knockdown of the human gene in a rodent expressing the human gene,e.g., an AAV-infected mouse expressing the human target gene (i.e.,xanthine dehydrogenase), e.g., when administered as a single dose, e.g.,at 3 mg/kg at the nadir of RNA expression. Knockdown of expression of anendogenous gene in a model animal system can also be determined, e.g.,after administration of a single dose at, e.g., 3 mg/kg at the nadir ofRNA expression. Such systems are useful when the nucleic acid sequenceof the human gene and the model animal gene are sufficiently close suchthat the human iRNA provides effective knockdown of the model animalgene. RNA expression in liver is determined using the PCR methodsprovided in Example 2.

Inhibition of the expression of a xanthine dehydrogenase gene may bemanifested by a reduction of the amount of mRNA expressed by a firstcell or group of cells (such cells may be present, for example, in asample derived from a subject) in which a xanthine dehydrogenase gene istranscribed and which has or have been treated (e.g., by contacting thecell or cells with an iRNA of the invention, or by administering an iRNAof the invention to a subject in which the cells are or were present)such that the expression of a xanthine dehydrogenase gene is inhibited,as compared to a second cell or group of cells substantially identicalto the first cell or group of cells but which has not or have not beenso treated (control cell(s) not treated with an iRNA or not treated withan iRNA targeted to the gene of interest). In some embodiments, theinhibition is assessed by the method provided in Example 2 using, e.g.,a 10 nM siRNA concentration in the species matched cell line andexpressing the level of mRNA in treated cells as a percentage of thelevel of mRNA in control cells, using the following formula:

${\frac{\left( {{mRNA}{in}{control}{cells}} \right) - \left( {{mRNA}{in}{treated}{cells}} \right)}{\left( {{mRNA}{in}{control}{cells}} \right)} \cdot 100}\%$

In other embodiments, inhibition of the expression of a xanthinedehydrogenase gene may be assessed in terms of a reduction of aparameter that is functionally linked to xanthine dehydrogenase geneexpression, e.g., xanthine dehydrogenase protein level in blood or serumfrom a subject. Xanthine dehydrogenase gene silencing may be determinedin any cell expressing xanthine dehydrogenase, either endogenous orheterologous from an expression construct, and by any assay known in theart.

Inhibition of the expression of a xanthine dehydrogenase protein may bemanifested by a reduction in the level of the xanthine dehydrogenaseprotein that is expressed by a cell or group of cells or in a subjectsample (e.g., the level of protein in a blood sample derived from asubject). As explained above, for the assessment of mRNA suppression,the inhibition of protein expression levels in a treated cell or groupof cells may similarly be expressed as a percentage of the level ofprotein in a control cell or group of cells, or the change in the levelof protein in a subject sample, e.g., blood or serum derived therefrom.

A control cell, a group of cells, or subject sample that may be used toassess the inhibition of the expression of a xanthine dehydrogenase geneincludes a cell, group of cells, or subject sample that has not yet beencontacted with an RNAi agent of the invention. For example, the controlcell, group of cells, or subject sample may be derived from anindividual subject (e.g., a human or animal subject) prior to treatmentof the subject with an RNAi agent or an appropriately matched populationcontrol.

The level of xanthine dehydrogenase mRNA that is expressed by a cell orgroup of cells may be determined using any method known in the art forassessing mRNA expression. In one embodiment, the level of expression ofxanthine dehydrogenase in a sample is determined by detecting atranscribed polynucleotide, or portion thereof, e.g., mRNA of thexanthine dehydrogenase gene. RNA may be extracted from cells using RNAextraction techniques including, for example, using acidphenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis),RNeasy™ RNA preparation kits (Qiagen®) or PAXgene™ (PreAnalytix™,Switzerland). Typical assay formats utilizing ribonucleic acidhybridization include nuclear run-on assays, RT-PCR, RNase protectionassays, northern blotting, in situ hybridization, and microarrayanalysis.

In some embodiments, the level of expression of xanthine dehydrogenaseis determined using a nucleic acid probe. The term “probe”, as usedherein, refers to any molecule that is capable of selectively binding toa specific xanthine dehydrogenase. Probes can be synthesized by one ofskill in the art, or derived from appropriate biological preparations.Probes may be specifically designed to be labeled. Examples of moleculesthat can be utilized as probes include, but are not limited to, RNA,DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or northern analyses,polymerase chain reaction (PCR) analyses and probe arrays. One methodfor the determination of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize to xanthinedehydrogenase mRNA. In one embodiment, the mRNA is immobilized on asolid surface and contacted with a probe, for example by running theisolated mRNA on an agarose gel and transferring the mRNA from the gelto a membrane, such as nitrocellulose. In an alternative embodiment, theprobe(s) are immobilized on a solid surface and the mRNA is contactedwith the probe(s), for example, in an Affymetrix® gene chip array. Askilled artisan can readily adapt known mRNA detection methods for usein determining the level of xanthine dehydrogenase mRNA.

An alternative method for determining the level of expression ofxanthine dehydrogenase in a sample involves the process of nucleic acidamplification or reverse transcriptase (to prepare cDNA) of for examplemRNA in the sample, e.g., by RT-PCR (the experimental embodiment setforth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction(Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustainedsequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardiet al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers. Inparticular aspects of the invention, the level of expression of XDH isdetermined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™System). In some embodiments, expression level is determined by themethod provided in Example 2 using, e.g., a 10 nM siRNA concentration,in the species matched cell line.

The expression levels of xanthine dehydrogenase mRNA may be monitoredusing a membrane blot (such as used in hybridization analysis such asnorthern, Southern, dot, and the like), or microwells, sample tubes,gels, beads or fibers (or any solid support comprising bound nucleicacids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195and 5,445,934, which are incorporated herein by reference. Thedetermination of xanthine dehydrogenase expression level may alsocomprise using nucleic acid probes in solution.

In some embodiments, the level of mRNA expression is assessed usingbranched DNA (bDNA) assays or real time PCR (qPCR). The use of thesemethods is described and exemplified in the Examples presented herein.In some embodiments, expression level is determined by the methodprovided in Example 2 using a 10 nM siRNA concentration in the speciesmatched cell line.

The level of XDH protein expression may be determined using any methodknown in the art for the measurement of protein levels. Such methodsinclude, for example, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions,absorption spectroscopy, a colorimetric assays, spectrophotometricassays, flow cytometry, immunodiffusion (single or double),immunoelectrophoresis, western blotting, radioimmunoassay (RIA),enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays,electrochemiluminescence assays, and the like.

In some embodiments, the efficacy of the methods of the invention areassessed by a decrease in XDH mRNA or protein level (e.g., in a liverbiopsy).

In some embodiments of the methods of the invention, the iRNA isadministered to a subject such that the iRNA is delivered to a specificsite within the subject. The inhibition of expression of xanthinedehydrogenase may be assessed using measurements of the level or changein the level of xanthine dehydrogenase mRNA or xanthine dehydrogenaseprotein in a sample derived from fluid or tissue from the specific sitewithin the subject (e.g., liver or blood).

As used herein, the terms detecting or determining a level of an analyteare understood to mean performing the steps to determine if a material,e.g., protein, RNA, is present. As used herein, methods of detecting ordetermining include detection or determination of an analyte level thatis below the level of detection for the method used.

VII. Prophylactic and Treatment Methods of the Invention

The present invention also provides methods of using an iRNA of theinvention or a composition containing an iRNA of the invention toinhibit expression of xanthine dehydrogenase, thereby preventing ortreating a xanthine dehydrogenase-associated disorder, e.g.,hyperuricemia, gout NAFLD, NASH, metabolic disorder, insulin resistance,cardiovascular disease, hypertension, type 2 diabetes, and conditionslinked to oxidative stress e.g., chronic low grade inflammation.

In one embodiment, the xanthine dehydrogenase-associate disease ishyperuricemia.

In another embodiment, the xanthine dehydrogenase-associate disease isgout.

In the methods of the invention the cell may be contacted with the siRNAin vitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may beany cell that expresses a xanthine dehydrogenase gene, e.g., a livercell, a brain cell, a gall bladder cell, a heart cell, or a kidney cell.In some embodiment, the cell is a liver cell. A cell suitable for use inthe methods of the invention may be a mammalian cell, e.g., a primatecell (such as a human cell, including human cell in a chimeric non-humananimal, or a non-human primate cell, e.g., a monkey cell or a chimpanzeecell), or a non-primate cell. In certain embodiments, the cell is ahuman cell, e.g., a human liver cell. In the methods of the invention,xanthine dehydrogenase expression is inhibited in the cell by at least50, 55, 60, 65, 70, 75, 80, 85, 90, or 95, or to a level below the levelof detection of the assay.

The in vivo methods of the invention may include administering to asubject a composition containing an iRNA, where the iRNA includes anucleotide sequence that is complementary to at least a part of an RNAtranscript of the xanthine dehydrogenase gene of the mammal to which theRNAi agent is to be administered. The composition can be administered byany means known in the art including, but not limited to oral,intraperitoneal, or parenteral routes, including intracranial (e.g.,intraventricular, intraparenchymal, and intrathecal), intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), nasal,rectal, and topical (including buccal and sublingual) administration. Incertain embodiments, the compositions are administered by intravenousinfusion or injection. In certain embodiments, the compositions areadministered by subcutaneous injection. In certain embodiments, thecompositions are administered by intramuscular injection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the iRNA in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof XDH, or a therapeutic or prophylactic effect. A depot injection mayalso provide more consistent serum concentrations. Depot injections mayinclude subcutaneous injections or intramuscular injections. In someembodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. In someembodiments, the infusion pump is a subcutaneous infusion pump. In otherembodiments, the pump is a surgically implanted pump that delivers theiRNA to the liver.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods forinhibiting the expression of a xanthine dehydrogenase gene in a mammal.The methods include administering to the mammal a composition comprisinga dsRNA that targets a xanthine dehydrogenase gene in a cell of themammal and maintaining the mammal for a time sufficient to obtaindegradation of the mRNA transcript of the xanthine dehydrogenase gene,thereby inhibiting expression of the xanthine dehydrogenase gene in thecell. Reduction in gene expression can be assessed by any methods knownin the art and by methods, e.g. qRT-PCR, described herein, e.g., inExample 2. Reduction in protein production can be assessed by anymethods known it the art, e.g. ELISA. In certain embodiments, a punctureliver biopsy sample serves as the tissue material for monitoring thereduction in the xanthine dehydrogenase gene or protein expression. Inother embodiments, a blood sample serves as the subject sample formonitoring the reduction in the xanthine dehydrogenase proteinexpression.

The present invention further provides methods of treatment in a subjectin need thereof, e.g., a subject diagnosed with a xanthinedehydrogenase-associated disorder, such as, hyperuricemia, gout NAFLD,NASH, metabolic disorder, insulin resistance, cardiovascular disease,hypertension, type 2 diabetes, and conditions linked to oxidative stresse.g., chronic low grade inflammation.

The present invention further provides methods of prophylaxis in asubject in need thereof. The treatment methods of the invention includeadministering an iRNA of the invention to a subject, e.g., a subjectthat would benefit from a reduction of xanthine dehydrogenaseexpression, in a prophylactically effective amount of an iRNA targetinga xanthine dehydrogenase gene or a pharmaceutical composition comprisingan iRNA targeting a xanthine dehydrogenase gene.

In one embodiment, the xanthine dehydrogenase-associate disease ishyperuricemia.

In another embodiment, the xanthine dehydrogenase-associate disease isgout.

An iRNA of the invention may be administered as a “free iRNA.” A freeiRNA is administered in the absence of a pharmaceutical composition. Thenaked iRNA may be in a suitable buffer solution. The buffer solution maycomprise acetate, citrate, prolamine, carbonate, or phosphate, or anycombination thereof. In one embodiment, the buffer solution is phosphatebuffered saline (PBS). The pH and osmolarity of the buffer solutioncontaining the iRNA can be adjusted such that it is suitable foradministering to a subject.

Alternatively, an iRNA of the invention may be administered as apharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from an inhibition of xanthine dehydrogenaseexpression are subjects susceptible to or diagnosed with anXDH-associated disorder, e.g., hyperuricemia or gout.

In an embodiment, the method includes administering a compositionfeatured herein such that expression of the target xanthinedehydrogenase gene is decreased, such as for about 1, 2, 3, 4, 5, 6,1-6, 1-3, or 3-6 months per dose. In certain embodiments, thecomposition is administered once every 3-6 months.

In some embodiments, the iRNAs useful for the methods and compositionsfeatured herein specifically target RNAs (primary or processed) of thetarget xanthine dehydrogenase gene. Compositions and methods forinhibiting the expression of these genes using iRNAs can be prepared andperformed as described herein.

Administration of the iRNA according to the methods of the invention mayresult prevention or treatment of a xanthine dehydrogenase-associateddisorder, e.g., hyperuricemia and gout.

Subjects can be administered a therapeutic amount of iRNA, such as about0.01 mg/kg to about 200 mg/kg. Subjects can be administered atherapeutic amount of iRNA, such as about 5 mg to about 1000 mg as afixed dose, regardless of body weight.

In some embodiments, the iRNA is administered subcutaneously, i.e., bysubcutaneous injection. One or more injections may be used to deliverthe desired dose of iRNA to a subject. The injections may be repeatedover a period of time.

The administration may be repeated on a regular basis. In certainembodiments, after an initial treatment regimen, the treatments can beadministered on a less frequent basis. A repeat-dose regimen may includeadministration of a therapeutic amount of iRNA on a regular basis, suchas once per month to once a year. In certain embodiments, the iRNA isadministered about once per month to about once every three months, orabout once every three months to about once every six months.

The invention further provides methods and uses of an iRNA agent or apharmaceutical composition thereof for treating a subject that wouldbenefit from reduction or inhibition of XDH gene expression, e.g., asubject having an XDH-associated disease, in combination with otherpharmaceuticals or other therapeutic methods, e.g., with knownpharmaceuticals or known therapeutic methods, such as, for example,those which are currently employed for treating these disorders.Exemplary pharmaceuticals include, for example, allopurinol, oxypurinol,Probenecid, Rasburicase, febuxostat, analgesic or anti-inflammatoryagents, e.g., NSAIDS.

Accordingly, in some aspects of the invention, the methods which includeeither a single iRNA agent of the invention, further includeadministering to the subject one or more additional therapeutic agents.The iRNA agent and an additional therapeutic agent or treatment may beadministered at the same time or in the same combination, e.g.,parenterally, or the additional therapeutic agent can be administered aspart of a separate composition or at separate times or by another methodknown in the art or described herein.

In one embodiment, an iRNA agent is administered in combination withallopurinol. In one embodiment, the iRNA agent is administered to thepatient, and then the additional therapeutic agent is administered tothe patient (or vice versa). In another embodiment, the iRNA agent andthe additional therapeutic agent are administered at the same time.

The iRNA agent and an additional therapeutic agent or treatment may beadministered at the same time or in the same combination, e.g.,parenterally, or the additional therapeutic agent can be administered aspart of a separate composition or at separate times or by another methodknown in the art or described herein.

VIII. Kits

In certain aspects, the instant disclosure provides kits that include asuitable container containing a pharmaceutical formulation of a siRNAcompound, e.g., a double-stranded siRNA compound, or ssiRNA compound,(e.g., a precursor, e.g., a larger siRNA compound which can be processedinto a ssiRNA compound, or a DNA which encodes an siRNA compound, e.g.,a double-stranded siRNA compound, or ssiRNA compound, or precursorthereof).

Such kits include one or more dsRNA agent(s) and instructions for use,e.g., instructions for administering a prophylactically ortherapeutically effective amount of a dsRNA agent(s). The dsRNA agentmay be in a vial or a pre-filled syringe. The kits may optionallyfurther comprise means for administering the dsRNA agent (e.g., aninjection device, such as a pre-filled syringe), or means for measuringthe inhibition of XDH (e.g., means for measuring the inhibition of XDHmRNA, XDH protein, or XDH activity). Such means for measuring theinhibition of XDH may comprise a means for obtaining a sample from asubject, such as, e.g., a plasma sample. The kits of the invention mayoptionally further comprise means for determining the therapeuticallyeffective or prophylactically effective amount.

In certain embodiments the individual components of the pharmaceuticalformulation may be provided in one container, e.g., a vial or apre-filled syringe. Alternatively, it may be desirable to provide thecomponents of the pharmaceutical formulation separately in two or morecontainers, e.g., one container for a siRNA compound preparation, and atleast another for a carrier compound. The kit may be packaged in anumber of different configurations such as one or more containers in asingle box. The different components can be combined, e.g., according toinstructions provided with the kit. The components can be combinedaccording to a method described herein, e.g., to prepare and administera pharmaceutical composition. The kit can also include a deliverydevice.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The entire contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the informal Sequence Listing and Figures,are hereby incorporated herein by reference.

EXAMPLES Example 1. iRNA Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Design

siRNAs targeting the xanthine dehydrogenase (XDH) gene, (human: NCBIrefseqID NM_000379; NCBI GeneID: 7498) were designed using custom R andPython scripts. The human NM_000379 REFSEQ mRNA, version 4, has a lengthof 5715 bases. Detailed lists of the unmodified XDH sense and antisensestrand nucleotide sequences are shown in Table 2. Detailed lists of themodified XDH sense and antisense strand nucleotide sequences are shownin Table 3.

It is to be understood that, throughout the application, a duplex namewithout a decimal is equivalent to a duplex name with a decimal whichmerely references the batch number of the duplex. For example, AD-959917is equivalent to AD-959917.1.

siRNA Synthesis

siRNAs were synthesized and annealed using routine methods known in theart.

Briefly, siRNA sequences were synthesized on a 1 μmol scale using aMermade 192 synthesizer (BioAutomation) with phosphoramidite chemistryon solid supports. The solid support was controlled pore glass (500-1000Å) loaded with a custom GalNAc ligand (3′-GalNAc conjugates), universalsolid support (AM Chemicals), or the first nucleotide of interest.Ancillary synthesis reagents and standard 2-cyanoethyl phosphoramiditemonomers (2′-deoxy-2′-fluoro, 2′-O-methyl, RNA, DNA) were obtained fromThermo-Fisher (Milwaukee, Wis.), Hongene (China), or Chemgenes(Wilmington, Mass., USA). Additional phosphoramidite monomers wereprocured from commercial suppliers, prepared in-house, or procured usingcustom synthesis from various CMOs. Phosphoramidites were prepared at aconcentration of 100 mM in either acetonitrile or 9:1 acetonitrile:DMFand were coupled using 5-Ethylthio-1H-tetrazole (ETT, 0.25 M inacetonitrile) with a reaction time of 400 s. Phosphorothioate linkageswere generated using a 100 mM solution of 3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes(Wilmington, Mass., USA)) in anhydrous acetonitrile/pyridine (9:1 v/v).Oxidation time was 5 minutes. All sequences were synthesized with finalremoval of the DMT group (“DMT-Off”).

Upon completion of the solid phase synthesis, solid-supportedoligoribonucleotides were treated with 300 μL of Methylamine (40%aqueous) at room temperature in 96 well plates for approximately 2 hoursto afford cleavage from the solid support and subsequent removal of alladditional base-labile protecting groups. For sequences containing anynatural ribonucleotide linkages (2′-OH) protected with a tert-butyldimethyl silyl (TBDMS) group, a second deprotection step was performedusing TEA.3HF (triethylamine trihydrofluoride). To each oligonucleotidesolution in aqueous methylamine was added 200 μL of dimethyl sulfoxide(DMSO) and 300 μL TEA.3HF and the solution was incubated forapproximately 30 mins at 60° C. After incubation, the plate was allowedto come to room temperature and crude oligonucleotides were precipitatedby the addition of 1 mL of 9:1 acetontrile:ethanol or 1:1ethanol:isopropanol. The plates were then centrifuged at 4° C. for 45mins and the supernatant carefully decanted with the aid of amultichannel pipette. The oligonucleotide pellet was resuspended in 20mM NaOAc and subsequently desalted using a HiTrap size exclusion column(5 mL, GE Healthcare) on an Agilent LC system equipped with anautosampler, UV detector, conductivity meter, and fraction collector.Desalted samples were collected in 96 well plates and then analyzed byLC-MS and UV spectrometry to confirm identity and quantify the amount ofmaterial, respectively.

Duplexing of single strands was performed on a Tecan liquid handlingrobot. Sense and antisense single strands were combined in an equimolarratio to a final concentration of 10 μM in 1× PBS in 96 well plates, theplate sealed, incubated at 100° C. for 10 minutes, and subsequentlyallowed to return slowly to room temperature over a period of 2-3 hours.The concentration and identity of each duplex was confirmed and thensubsequently utilized for in vitro screening assays.

Example 2. In Vitro Screening Methods Cell Culture and Free UptakeExperiments

Primary mouse hepatocytes (PMH) or primary human hepatocytes (PHH) werefreshly isolated less than 1 hour prior to transfection and grown inprimary hepatocyte media.

Free uptake experiments were performed by adding 2.5 μl of siRNAduplexes in PBS per well into a 96 well plate. Complete growth media(47.5 μl) containing about 1.5×10⁴ PMH or PHH was then added to thesiRNA. Cells were incubated for 48 hours prior to RNA purification andRT-qPCR. Single dose experiments in PHH were performed at 500 nM, 100nM, 10 nM and/or 0.1 nM in PHH and in PMH at 500 nM, 100 nM, nM, and 1nM final duplex concentration.

Total RNA isolation was performed using DYNABEADS. Briefly, cells werelysed in 10 μl of Lysis/Binding Buffer containing 3 μL of beads per welland mixed for 10 minutes on an electrostatic shaker. The washing stepswere automated on a Biotek EL406, using a magnetic plate support. Beadswere washed (in 3 μL) once in Buffer A, once in Buffer B, and twice inBuffer E, with aspiration steps in between. Following a finalaspiration, complete 12 μL RT mixture was added to each well, asdescribed below.

For cDNA synthesis, a master mix of 1.5 μl 10× Buffer, 0.6 μl 10× dNTPs,1.5 μl Random primers, 0.75 μl Reverse Transcriptase, 0.75 μl RNaseinhibitor and 9.9 μl of H₂O per reaction were added per well. Plateswere sealed, agitated for 10 minutes on an electrostatic shaker, andthen incubated at 37 degrees C. for 2 hours. Following this, the plateswere agitated at 80 degrees C. for 8 minutes.

For RT-qPCR, two microlitre (μl) of cDNA were added to a master mixcontaining 0.5 μl of human GAPDH TaqMan Probe (4326317E), 0.5 μl humanAPOC3, 2 μl nuclease-free water and 5 μl Lightcycler 480 probe mastermix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat#04887301001). Real time PCR was done in a LightCycler480 Real Time PCRsystem (Roche).

To calculate relative fold change, data were analyzed using the ΔΔCtmethod and normalized to assays performed with cells transfected with 10nM AD-1955, or mock transfected cells. IC₅₀s were calculated using a 4parameter fit model using XLFit and normalized to cells transfected withAD-1955 or mock-transfected. The sense and antisense sequences ofAD-1955 are: sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 21) andantisense UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 22).

The results of the free uptake experiments of the dsRNA agents listed inTables 2 and 3 in PHH are shown in Table 4 and the results of the freeuptake experiments of the dsRNA agents listed in Tables 2 and 3 in PMHare shown in Table 5.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds; and it is understood that when the nucleotidecontains a 2′-fluoro modification, then the fluoro replaces the hydroxyat that position in the parent nucleotide (i.e., it is a 2′-deoxy-2′-fluoronucleotide). Abbreviation Nucleotide(s) A Adenosine-3′-phosphateAb beta-L-adenosine-3′-phosphate Absbeta-L-adenosine-3′-phosphorothioate Af 2′-fluoroadenosine-3′-phosphateAfs 2′-fluoroadenosine-3′-phosphorothioate Asadenosine-3′-phosphorothioate C cytidine-3′-phosphate Cbbeta-L-cytidine-3′-phosphate Cbs beta-L-cytidine-3′-phosphorothioate Cf2′-fluorocytidine-3′-phosphate Cfs 2′-fluorocytidine-3′-phosphorothioateCs cytidine-3′-phosphorothioate G guanosine-3′-phosphate Gbbeta-L-guanosine-3′-phosphate Gbs beta-L-guanosine-3′-phosphorothioateGf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioateUs uridine-3′-phosphorothioate N any nucleotide, modified or unmodifieda 2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate g2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L10N-(cholesterylcarboxamidocaproyl)-4-hydroxyprolinol (Hyp-C6-Chol) L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol(Hyp-(GalNAc-alkyl)3)

Y34 2-hydroxymethyl-tetrahydrofurane-4-methoxy-3-phosphate (abasic2′-OMe furanose) Y44 inverted abasic DNA(2-hydroxymethyl-tetrahydrofurane-5-phosphate) (Agn) Adenosine-glycolnucleic acid (GNA) (Cgn) Cytidine-glycol nucleic acid (GNA) (Ggn)Guanosine-glycol nucleic acid (GNA) (Tgn) Thymidine-glycol nucleic acid(GNA) S-Isomer P Phosphate VP Vinyl-phosphonate dA2′-deoxyadenosine-3′-phosphate dAs 2′-deoxyadenosine-3′-phosphorothioate dC 2′-deoxy cytidine-3′-phosphate dCs2′-deoxy cytidine-3′-phosphorothioate dG 2′-deoxy guanosine-3′-phosphatedGs 2′-deoxy guanosine-3′-phosphorothioate dT2'-deoxythimidine-3′-phosphate dTs 2′-deoxythimidine-3′-phosphorothioatedU 2′-deoxyuridine dUs 2′-deoxyuridine-3′-phosphorothioate (C2p)cytidine-2′-phosphate (G2p) guanosine-2′-phosphate (U2p)uridine-2′-phosphate (A2p) adenosine-2′-phosphate (Ahd)2′-O-hexadecyl-adenosine-3′-phosphate (Chd)2′-O-hexadecyl-cytidine-3′-phosphate (Ghd)2′-O-hexadecyl-guanosine-3′-phosphate (Uhd)2′-O-hexadecyl-uridine-3′-phosphate (Aams)2′-O-(N-methylacetamide)adenosine-3′-phosphorothioate (Gams)2′-O-(N-methylacetamide)guanosine-3′-phosphorothioate (Tams)2′-O-(N-methylacetamide)thymidine-3′-phosphorothioate

TABLE 2 Unmodified Sense and Antisense Strand Sequences ofXanthine Dehydrogenase dsRNA Agents SEQ SEQ Sense Sequence ID Range inAntisense Sequence ID Range in Duplex Name 5′ to 3′ NO: NM_000379.45′ to 3′ NO: NM_000379.4 AD-1135979.1 UACCUGCCAGUGUCUCUUAGU 23 17-37ACUAAGAGACACUGGCAGGUAGU 382 15-37 AD-1135980.1 ACCUGCCAGUGUCUCUUAGGU 2418-38 ACCUAAGAGACACUGGCAGGUAG 383 16-38 AD-1135981.1CCUGCCAGUGUCUCUUAGGAU 25 19-39 AUCCUAAGAGACACUGGCAGGUA 384 17-39AD-1135982.1 UGCCAGUGUCUCUUAGGAGUU 26 21-41 AACUCCUAAGAGACACUGGCAGG 38519-41 AD-1135983.1 GCCAGUGUCUCUUAGGAGUGU 27 22-42ACACTCCUAAGAGACACUGGCAG 386 20-42 AD-1135984.1 CCAGUGUCUCUUAGGAGUGAU 2823-43 AUCACUCCUAAGAGACACUGGCA 387 21-43 AD-1135985.1CAGUGUCUCUUAGGAGUGAGU 29 24-44 ACUCACUCCUAAGAGACACUGGC 388 22-44AD-1135986.1 AGUGUCUCUUAGGAGUGAGGU 30 25-45 ACCUCACUCCUAAGAGACACUGG 38923-45 AD-1135987.1 UGGAGAAAAAUGCAGAUCCAU 31 123-143AUGGAUCUGCAUUUUUCUCCACC 390 121-143 AD-1135988.1 CCAGAGACAACCCUUUUGGCU32 140-160 AGCCAAAAGGGUUGUCUCUGGAU 391 138-160 AD-1135989.1AGAGACAACCCUUUUGGCCUU 33 142-162 AAGGCCAAAAGGGUUGUCUCUGG 392 140-162AD-1135990.1 GCACAGUGAUGCUCUCCAAGU 34 228-248 ACUUGGAGAGCAUCACUGUGCAA393 226-248 AD-1135991.1 CACAGUGAUGCUCUCCAAGUU 35 229-249AACUTGGAGAGCAUCACUGUGCA 394 227-249 AD-1135992.1 GAUGCUCUCCAAGUAUGAUCU36 235-255 AGAUCAUACUUGGAGAGCAUCAC 395 233-255 AD-1135993.1AUGCUCUCCAAGUAUGAUCGU 37 236-256 ACGAUCAUACUUGGAGAGCAUCA 396 234-256AD-1135994.1 UGCUCUCCAAGUAUGAUCGUU 38 237-257 AACGAUCAUACUUGGAGAGCAUC397 235-257 AD-1135995.1 GCUCUCCAAGUAUGAUCGUCU 39 238-258AGACGAUCAUACUUGGAGAGCAU 398 236-258 AD-1135996.1 CUCUCCAAGUAUGAUCGUCUU40 239-259 AAGACGAUCAUACUUGGAGAGCA 399 237-259 AD-1135997.1GAUCGUCUGCAGAACAAGAUU 41 251-271 AAUCTUGUUCUGCAGACGAUCAU 400 249-271AD-1135998.1 CGUCUGCAGAACAAGAUCGUU 42 254-274 AACGAUCUUGUUCUGCAGACGAU401 252-274 AD-1135999.1 GUCUGCAGAACAAGAUCGUCU 43 255-275AGACGAUCUUGUUCUGCAGACGA 402 253-275 AD-1136000.1 UCUGCAGAACAAGAUCGUCCU44 256-276 AGGACGAUCUUGUUCUGCAGACG 403 254-276 AD-1136001.1GCAGAACAAGAUCGUCCACUU 45 259-279 AAGUGGACGAUCUUGUUCUGCAG 404 257-279AD-1136002.1 CAGAACAAGAUCGUCCACUUU 46 260-280 AAAGTGGACGAUCUUGUUCUGCA405 258-280 AD-1136003.1 GAACAAGAUCGUCCACUUUUU 47 262-282AAAAAGTGGACGAUCUUGUUCUG 406 260-282 AD-1136004.1 AACAAGAUCGUCCACUUUUCU48 263-283 AGAAAAGUGGACGAUCUUGUUCU 407 261-283 AD-1136005.1ACAAGAUCGUCCACUUUUCUU 49 264-284 AAGAAAAGUGGACGAUCUUGUUC 408 262-284AD-1136006.1 CAAGAUCGUCCACUUUUCUGU 50 265-285 ACAGAAAAGUGGACGAUCUUGUU409 263-285 AD-1136007.1 AAGAUCGUCCACUUUUCUGCU 51 266-286AGCAGAAAAGUGGACGAUCUUGU 410 264-286 AD-1136008.1 CGUCCACUUUUCUGCCAAUGU52 271-291 ACAUTGGCAGAAAAGUGGACGAU 411 269-291 AD-1136009.1GUCCACUUUUCUGCCAAUGCU 53 272-292 AGCATUGGCAGAAAAGUGGACGA 412 270-292AD-1136010.1 UGCUCCUUGCACCAUGUUGCU 54 308-328 AGCAACAUGGUGCAAGGAGCAGA413 306-328 AD-1136011.1 UCCUUGCACCAUGUUGCAGUU 55 311-331AACUGCAACAUGGUGCAAGGAGC 414 309-331 AD-1136012.1 UUGCACCAUGUUGCAGUGACU56 314-334 AGUCACTGCAACAUGGUGCAAGG 415 312-334 AD-1136013.1CACCAUGUUGCAGUGACAACU 57 317-337 AGUUGUCACUGCAACAUGGUGCA 416 315-337AD-1136014.1 ACCAUGUUGCAGUGACAACUU 58 318-338 AAGUTGTCACUGCAACAUGGUGC417 316-338 AD-1136015.1 AGGAGAGAAUUGCCAAAAGCU 59 381-401AGCUTUTGGCAAUUCUCUCCUGC 418 379-401 AD-1136016.1 GGAGAGAAUUGCCAAAAGCCU60 382-402 AGGCTUTUGGCAAUUCUCUCCUG 419 380-402 AD-1136017.1UGGCAUCGUCAUGAGUAUGUU 61 430-450 AACAUACUCAUGACGAUGCCAGG 420 428-450AD-1136018.1 GGCAUCGUCAUGAGUAUGUAU 62 431-451 AUACAUACUCAUGACGAUGCCAG421 429-451 AD-1136019.1 GCAUCGUCAUGAGUAUGUACU 63 432-452AGUACAUACUCAUGACGAUGCCA 422 430-452 AD-1136020.1 CAUCGUCAUGAGUAUGUACAU64 433-453 AUGUACAUACUCAUGACGAUGCC 423 431-453 AD-1136021.1AUCGUCAUGAGUAUGUACACU 65 434-454 AGUGTACAUACUCAUGACGAUGC 424 432-454AD-1136022.1 CGUCAUGAGUAUGUACACACU 66 436-456 AGUGTGTACAUACUCAUGACGAU425 434-456 AD-1136023.1 GUCAUGAGUAUGUACACACUU 67 437-457AAGUGUGUACAUACUCAUGACGA 426 435-457 AD-1136024.1 UCAUGAGUAUGUACACACUGU68 438-458 ACAGTGTGUACAUACUCAUGACG 427 436-458 AD-1136025.1AAUGCCUUCCAAGGAAAUCUU 69 497-517 AAGATUTCCUUGGAAGGCAUUCU 428 495-517AD-1136026.1 AUGCCUUCCAAGGAAAUCUGU 70 498-518 ACAGAUUUCCUUGGAAGGCAUUC429 496-518 AD-1136027.1 UGCCUUCCAAGGAAAUCUGUU 71 499-519AACAGAUUUCCUUGGAAGGCAUU 430 497-519 AD-1136028.1 GCCUUCCAAGGAAAUCUGUGU72 500-520 ACACAGAUUUCCUUGGAAGGCAU 431 498-520 AD-1136029.1CCUUCCAAGGAAAUCUGUGCU 73 501-521 AGCACAGAUUUCCUUGGAAGGCA 432 499-521AD-1136030.1 GAGAUGAAGUUCAAGAAUAUU 74 875-895 AAUATUCUUGAACUUCAUCUCAA433 873-895 AD-1136031.1 GAUGAAGUUCAAGAAUAUGCU 75 877-897AGCAUAUUCUUGAACUUCAUCUC 434 875-897 AD-1136032.1 AAGUUCAAGAAUAUGCUGUUU76 881-901 AAACAGCAUAUUCUUGAACUUCA 435 879-901 AD-1136033.1AGUUCAAGAAUAUGCUGUUUU 77 882-902 AAAACAGCAUAUUCUUGAACUUC 436 880-902AD-1136034.1 GUUCAAGAAUAUGCUGUUUCU 78 883-903 AGAAACAGCAUAUUCUUGAACUU437 881-903 AD-1136035.1 UUCAAGAAUAUGCUGUUUCCU 79 884-904AGGAAACAGCAUAUUCUUGAACU 438 882-904 AD-1136036.1 AAGAAUAUGCUGUUUCCUAUU80 887-907 AAUAGGAAACAGCAUAUUCUUGA 439 885-907 AD-1136037.1GGGAGUAUUUCUCAGCAUUCU 81 1320-1340 AGAATGCUGAGAAAUACUCCCCC 440 1318-1340AD-1136038.1 GGAGUAUUUCUCAGCAUUCAU 82 1321-1341 AUGAAUGCUGAGAAAUACUCCCC441 1319-1341 AD-1136039.1 GAGUAUUUCUCAGCAUUCAAU 83 1322-1342AUUGAAUGCUGAGAAAUACUCCC 442 1320-1342 AD-1136040.1 AGUAUUUCUCAGCAUUCAAGU84 1323-1343 ACUUGAAUGCUGAGAAAUACUCC 443 1321-1343 AD-1136041.1GUAUUUCUCAGCAUUCAAGCU 85 1324-1344 AGCUTGAAUGCUGAGAAAUACUC 444 1322-1344AD-1136042.1 GUGGCAUGAGAGUUUUAUUCU 86 1383-1403 AGAAUAAAACUCUCAUGCCACUG445 1381-1403 AD-1136043.1 GGCAUGAGAGUUUUAUUCAAU 87 1385-1405AUUGAAUAAAACUCUCAUGCCAC 446 1383-1405 AD-1136044.1 CAUGAGAGUUUUAUUCAAGCU88 1387-1407 AGCUTGAAUAAAACUCUCAUGCC 447 1385-1407 AD-1136045.1CCUCACCCUCAGCUUCUUCUU 89 1606-1626 AAGAAGAAGCUGAGGGUGAGGGU 448 1604-1626AD-1136046.1 UCACCCUCAGCUUCUUCUUCU 90 1608-1628 AGAAGAAGAAGCUGAGGGUGAGG449 1606-1628 AD-1136047.1 UCUUCAAGUUCUACCUGACAU 91 1623-1643AUGUCAGGUAGAACUUGAAGAAG 450 1621-1643 AD-1136048.1 UUUCGCCAGUGCAACUUUACU92 1702-1722 AGUAAAGUUGCACUGGCGAAAGU 451 1700-1722 AD-1136049.1UUCGCCAGUGCAACUUUACUU 93 1703-1723 AAGUAAAGUUGCACUGGCGAAAG 452 1701-1723AD-1136050.1 UUCCAGGGUUUGUUUGUUUCU 94 1962-1982 AGAAACAAACAAACCCUGGAACC453 1960-1982 AD-1136051.1 CAGGGUUUGUUUGUUUCAUUU 95 1965-1985AAAUGAAACAAACAAACCCUGGA 454 1963-1985 AD-1136052.1 GGGUUUGUUUGUUUCAUUUCU96 1967-1987 AGAAAUGAAACAAACAAACCCUG 455 1965-1987 AD-1136053.1GGUUUGUUUGUUUCAUUUCCU 97 1968-1988 AGGAAAUGAAACAAACAAACCCU 456 1966-1988AD-1136054.1 UUGUUUGUUUCAUUUCCGCUU 98 1971-1991 AAGCGGAAAUGAAACAAACAAAC457 1969-1991 AD-1136055.1 UUCCUGGGAGUAACAUAACUU 99 1998-2018AAGUUAUGUUACUCCCAGGAACA 458 1996-2018 AD-1136056.1 GGGAGUAACAUAACUGGAAUU100 2003-2023 AAUUCCAGUUAUGUUACUCCCAG 459 2001-2023 AD-1136057.1UAACAUAACUGGAAUUUGUAU 101 2008-2028 AUACAAAUUCCAGUUAUGUUACU 4602006-2028 AD-1136058.1 AACAUAACUGGAAUUUGUAAU 102 2009-2029AUUACAAAUUCCAGUUAUGUUAC 461 2007-2029 AD-1136059.1 CGAAGGAUAAGGUUACUUGUU103 2046-2066 AACAAGUAACCUUAUCCUUCGCA 462 2044-2066 AD-1136060.1GAAGGAUAAGGUUACUUGUGU 104 2047-2067 ACACAAGUAACCUUAUCCUUCGC 4632045-2067 AD-1136061.1 GGGUGAAAAUCACCUAUGAAU 105 2130-2150AUUCAUAGGUGAUUUUCACCCCU 464 2128-2150 AD-1136062.1 AAUCACCUAUGAAGAACUACU106 2137-2157 AGUAGUTCUUCAUAGGUGAUUUU 465 2135-2157 AD-1136063.1AUCACCUAUGAAGAACUACCU 107 2138-2158 AGGUAGTUCUUCAUAGGUGAUUU 4662136-2158 AD-1136064.1 UACCAGCCAUUAUCACAAUUU 108 2154-2174AAAUTGTGAUAAUGGCUGGUAGU 467 2152-2174 AD-1136065.1 GAACAACUCCUUUUAUGGACU109 2188-2208 AGUCCAUAAAAGGAGUUGUUCUU 468 2186-2208 AD-1136066.1GGCCAAGAGCACUUCUACCUU 110 2291-2311 AAGGTAGAAGUGCUCUUGGCCAC 4692289-2311 AD-1136067.1 GAGCACUUCUACCUGGAGACU ill 2297-2317AGUCTCCAGGUAGAAGUGCUCUU 470 2295-2317 AD-1136068.1 GCACUUCUACCUGGAGACUCU112 2299-2319 AGAGTCTCCAGGUAGAAGUGCUC 471 2297-2319 AD-1136069.1CACUUCUACCUGGAGACUCAU 113 2300-2320 AUGAGUCUCCAGGUAGAAGUGCU 4722298-2320 AD-1136070.1 UCACUGCACCAUUGCUGUUCU 114 2317-2337AGAACAGCAAUGGUGCAGUGAGU 473 2315-2337 AD-1136071.1 CACUGCACCAUUGCUGUUCCU115 2318-2338 AGGAACAGCAAUGGUGCAGUGAG 474 2316-2338 AD-1136072.1CCAUUGCUGUUCCAAAAGGCU 116 2325-2345 AGCCUUUUGGAACAGCAAUGGUG 4752323-2345 AD-1136073.1 AGCUCUUUGUGUCUACACAGU 117 2361-2381ACUGTGTAGACACAAAGAGCUCC 476 2359-2381 AD-1136074.1 GCUCUUUGUGUCUACACAGAU118 2362-2382 AUCUGUGUAGACACAAAGAGCUC 477 2360-2382 AD-1136075.1CUCUUUGUGUCUACACAGAAU 119 2363-2383 AUUCTGTGUAGACACAAAGAGCU 4782361-2383 AD-1136076.1 UCUUUGUGUCUACACAGAACU 120 2364-2384AGUUCUGUGUAGACACAAAGAGC 479 2362-2384 AD-1136077.1 CUUUGUGUCUACACAGAACAU121 2365-2385 AUGUTCTGUGUAGACACAAAGAG 480 2363-2385 AD-1136078.1UUUGUGUCUACACAGAACACU 122 2366-2386 AGUGTUCUGUGUAGACACAAAGA 4812364-2386 AD-1136079.1 ACACAGAACACCAUGAAGACU 123 2375-2395AGUCTUCAUGGUGUUCUGUGUAG 482 2373-2395 AD-1136080.1 GAGCUUUGUUGCAAAAAUGUU124 2398-2418 AACAUUUUUGCAACAAAGCUCUG 483 2396-2418 AD-1136081.1AGCUUUGUUGCAAAAAUGUUU 125 2399-2419 AAACAUUUUUGCAACAAAGCUCU 4842397-2419 AD-1136082.1 AGGAUCUCUCUCAGAGUAUUU 126 2691-2711AAAUACTCUGAGAGAGAUCCUGG 485 2689-2711 AD-1136083.1 GGAUCUCUCUCAGAGUAUUAU127 2692-2712 AUAATACUCUGAGAGAGAUCCUG 486 2690-2712 AD-1136084.1GAUCUCUCUCAGAGUAUUAUU 128 2693-2713 AAUAAUACUCUGAGAGAGAUCCU 4872691-2713 AD-1136085.1 AUCUCUCUCAGAGUAUUAUGU 129 2694-2714ACAUAAUACUCUGAGAGAGAUCC 488 2692-2714 AD-1136086.1 UCUCUCUCAGAGUAUUAUGGU130 2695-2715 ACCAUAAUACUCUGAGAGAGAUC 489 2693-2715 AD-1136087.1CUCUCUCAGAGUAUUAUGGAU 131 2696-2716 AUCCAUAAUACUCUGAGAGAGAU 4902694-2716 AD-1136088.1 UCUCUCAGAGUAUUAUGGAAU 132 2697-2717AUUCCAUAAUACUCUGAGAGAGA 491 2695-2717 AD-1136089.1 CUCUCAGAGUAUUAUGGAACU133 2698-2718 AGUUCCAUAAUACUCUGAGAGAG 492 2696-2718 AD-1136090.1UCUCAGAGUAUUAUGGAACGU 134 2699-2719 ACGUUCCAUAAUACUCUGAGAGA 4932697-2719 AD-1136091.1 UCAGAGUAUUAUGGAACGAGU 135 2701-2721ACUCGUUCCAUAAUACUCUGAGA 494 2699-2721 AD-1136092.1 CAGAGUAUUAUGGAACGAGCU136 2702-2722 AGCUCGUUCCAUAAUACUCUGAG 495 2700-2722 AD-1136093.1GCUGUGCAAAACCAACCUUCU 137 2776-2796 AGAAGGTUGGUUUUGCACAGCCG 4962774-2796 AD-1136094.1 CUGUGCAAAACCAACCUUCCU 138 2777-2797AGGAAGGUUGGUUUUGCACAGCC 497 2775-2797 AD-1136095.1 CCUGACACACUUCAACCAGAU139 2932-2952 AUCUGGTUGAAGUGUGUCAGGUC 498 2930-2952 AD-1136096.1UGACACACUUCAACCAGAAGU 140 2934-2954 ACUUCUGGUUGAAGUGUGUCAGG 4992932-2954 AD-1136097.1 GACACACUUCAACCAGAAGCU 141 2935-2955AGCUTCTGGUUGAAGUGUGUCAG 500 2933-2955 AD-1136098.1 ACACUUCAACCAGAAGCUUGU142 2938-2958 ACAAGCUUCUGGUUGAAGUGUGU 501 2936-2958 AD-1136099.1AUGCCUAGCAAGCUCUCAGUU 143 2989-3009 AACUGAGAGCUUGCUAGGCAUUC 5022987-3009 AD-1136100.1 CCUAGCAAGCUCUCAGUAUCU 144 2992-3012AGAUACTGAGAGCUUGCUAGGCA 503 2990-3012 AD-1136101.1 CUAGCAAGCUCUCAGUAUCAU145 2993-3013 AUGATACUGAGAGCUUGCUAGGC 504 2991-3013 AD-1136102.1UAGCAAGCUCUCAGUAUCAUU 146 2994-3014 AAUGAUACUGAGAGCUUGCUAGG 5052992-3014 AD-1136103.1 AGCAAGCUCUCAGUAUCAUGU 147 2995-3015ACAUGAUACUGAGAGCUUGCUAG 506 2993-3015 AD-1136104.1 CAAGCUCUCAGUAUCAUGCUU148 2997-3017 AAGCAUGAUACUGAGAGCUUGCU 507 2995-3017 AD-1136105.1CUCGGAAGAGUGAGGUUGACU 149 3015-3035 AGUCAACCUCACUCUUCCGAGCA 5083013-3035 AD-1136106.1 AAGGAGAAUUGUUGGAAAAAU 150 3044-3064AUUUTUCCAACAAUUCUCCUUGU 509 3042-3064 AD-1136107.1 CACCAAGUUUGGAAUAAGCUU151 3085-3105 AAGCUUAUUCCAAACUUGGUGGG 510 3083-3105 AD-1136108.1ACCAAGUUUGGAAUAAGCUUU 152 3089-3109 AAAGCUUAUUCCAAACUUGGUGG 5113087-3109 AD-1136109.1 AGUUCCUUUUCUGAAUCAGGU 153 3109-3129ACCUGAUUCAGAAAAGGAACUGU 512 3107-3129 AD-1136110.1 GUUCCUUUUCUGAAUCAGGCU154 3110-3130 AGCCTGAUUCAGAAAAGGAACUG 513 3108-3130 AD-1136111.1UUCCUUUUCUGAAUCAGGCAU 155 3111-3131 AUGCCUGAUUCAGAAAAGGAACU 5143109-3131 AD-1136112.1 UACUUCAUGUGUACACAGAUU 156 3138-3158AAUCTGTGUACACAUGAAGUAGG 515 3136-3158 AD-1136114.1 CAAGGCCUUCAUACCAAAAUU157 3197-3217 AAUUTUGGUAUGAAGGCCUUGGC 516 3195-3217 AD-1136115.1AAGGCCUUCAUACCAAAAUGU 158 3198-3218 ACAUUUUGGUAUGAAGGCCUUGG 5173196-3218 AD-1136116.1 GCCUUCAUACCAAAAUGGUCU 159 3201-3221AGACCAUUUUGGUAUGAAGGCCU 518 3199-3221 AD-1136117.1 CACCUCUAAGAUUUAUAUCAU160 3250-3270 AUGAUAUAAAUCUUAGAGGUGGG 519 3248-3270 AD-1136118.1ACCUCUAAGAUUUAUAUCAGU 161 3251-3271 ACUGAUAUAAAUCUUAGAGGUGG 5203249-3271 AD-1136119.1 GCGAGACAAGCACUAACACUU 162 3270-3290AAGUGUTAGUGCUUGUCUCGCUG 521 3268-3290 AD-1136120.1 CGAGACAAGCACUAACACUGU163 3271-3291 ACAGTGTUAGUGCUUGUCUCGCU 522 3269-3291 AD-1136121.1GAGACAAGCACUAACACUGUU 164 3272-3292 AACAGUGUUAGUGCUUGUCUCGC 5233270-3292 AD-1136122.1 AGACAAGCACUAACACUGUGU 165 3273-3293ACACAGTGUUAGUGCUUGUCUCG 524 3271-3293 AD-1136123.1 CGGCUUGUCAGACCAUCUUGU166 3354-3374 ACAAGAUGGUCUGACAAGCCGCA 525 3352-3374 AD-1136124.1GGCUUGUCAGACCAUCUUGAU 167 3355-3375 AUCAAGAUGGUCUGACAAGCCGC 5263353-3375 AD-1136125.1 GCUUGUCAGACCAUCUUGAAU 168 3356-3376AUUCAAGAUGGUCUGACAAGCCG 527 3354-3376 AD-1136126.1 CUUGUCAGACCAUCUUGAAAU169 3357-3377 AUUUCAAGAUGGUCUGACAAGCC 528 3355-3377 AD-1136127.1UUGUCAGACCAUCUUGAAAAU 170 3358-3378 AUUUTCAAGAUGGUCUGACAAGC 5293356-3378 AD-1136128.1 UGUCAGACCAUCUUGAAAAGU 171 3359-3379ACUUUUCAAGAUGGUCUGACAAG 530 3357-3379 AD-1136129.1 GUCAGACCAUCUUGAAAAGGU172 3360-3380 ACCUUUUCAAGAUGGUCUGACAA 531 3358-3380 AD-1136130.1UCAGACCAUCUUGAAAAGGCU 173 3361-3381 AGCCUUUUCAAGAUGGUCUGACA 5323359-3381 AD-1136131.1 ACCCUACAAGAAGAAGAAUCU 174 3385-3405AGAUTCTUCUUCUUGUAGGGUUC 533 3383-3405 AD-1136132.1 CUGCCACUGGGUUUUAUAGAU175 3462-3482 AUCUAUAAAACCCAGUGGCAGAC 534 3460-3482 AD-1136133.1UGCCACUGGGUUUUAUAGAAU 176 3463-3483 AUUCUAUAAAACCCAGUGGCAGA 5353461-3483 AD-1136134.1 CACUGGGUUUUAUAGAACACU 177 3466-3486AGUGTUCUAUAAAACCCAGUGGC 536 3464-3486 AD-1136135.1 ACUGGGUUUUAUAGAACACCU178 3467-3487 AGGUGUTCUAUAAAACCCAGUGG 537 3465-3487 AD-1136136.1GGGUUUUAUAGAACACCCAAU 179 3470-3490 AUUGGGTGUUCUAUAAAACCCAG 5383468-3490 AD-1136137.1 GGUUUUAUAGAACACCCAAUU 180 3471-3491AAUUGGGUGUUCUAUAAAACCCA 539 3469-3491 AD-1136138.1 UGGGCUACAGCUUUGAGACUU181 3492-3512 AAGUCUCAAAGCUGUAGCCCAGA 540 3490-3512 AD-1136139.1GGGCUACAGCUUUGAGACUAU 182 3493-3513 AUAGTCTCAAAGCUGUAGCCCAG 5413491-3513 AD-1136140.1 GCUACAGCUUUGAGACUAACU 183 3495-3515AGUUAGTCUCAAAGCUGUAGCCC 542 3493-3515 AD-1136141.1 CUACAGCUUUGAGACUAACUU184 3496-3516 AAGUTAGUCUCAAAGCUGUAGCC 543 3494-3516 AD-1136142.1UACAGCUUUGAGACUAACUCU 185 3497-3517 AGAGTUAGUCUCAAAGCUGUAGC 5443495-3517 AD-1136143.1 ACAGCUUUGAGACUAACUCAU 186 3498-3518AUGAGUTAGUCUCAAAGCUGUAG 545 3496-3518 AD-1136144.1 CAGCUUUGAGACUAACUCAGU187 3499-3519 ACUGAGUUAGUCUCAAAGCUGUA 546 3497-3519 AD-1136145.1AGCUUUGAGACUAACUCAGGU 188 3500-3520 ACCUGAGUUAGUCUCAAAGCUGU 5473498-3520 AD-1136146.1 CUUCCACUACUUCAGCUAUGU 189 3526-3546ACAUAGCUGAAGUAGUGGAAGGG 548 3524-3546 AD-1136147.1 UUCCACUACUUCAGCUAUGGU190 3527-3547 ACCAUAGCUGAAGUAGUGGAAGG 549 3525-3547 AD-1136148.1GUGGCUUGCUCUGAAGUAGAU 191 3548-3568 AUCUACTUCAGAGCAAGCCACCC 5503546-3568 AD-1136149.1 UGGCUUGCUCUGAAGUAGAAU 192 3549-3569AUUCTACUUCAGAGCAAGCCACC 551 3547-3569 AD-1136150.1 GGCUUGCUCUGAAGUAGAAAU193 3550-3570 AUUUCUACUUCAGAGCAAGCCAC 552 3548-3570 AD-1136151.1GCUUGCUCUGAAGUAGAAAUU 194 3551-3571 AAUUTCTACUUCAGAGCAAGCCA 5533549-3571 AD-1136152.1 CCUAACAGGAGAUCAUAAGAU 195 3577-3597AUCUUAUGAUCUCCUGUUAGGCA 554 3575-3597 AD-1136153.1 CUAACAGGAGAUCAUAAGAAU196 3578-3598 AUUCTUAUGAUCUCCUGUUAGGC 555 3576-3598 AD-1136154.1UAACAGGAGAUCAUAAGAACU 197 3579-3599 AGUUCUUAUGAUCUCCUGUUAGG 5563577-3599 AD-1136155.1 AACAGGAGAUCAUAAGAACCU 198 3580-3600AGGUTCTUAUGAUCUCCUGUUAG 557 3578-3600 AD-1136156.1 CCUCCGCACAGAUAUUGUCAU199 3598-3618 AUGACAAUAUCUGUGCGGAGGUU 558 3596-3618 AD-1136157.1CUCCGCACAGAUAUUGUCAUU 200 3599-3619 AAUGACAAUAUCUGUGCGGAGGU 5593597-3619 AD-1136158.1 UUGGCUCCAGUCUAAACCCUU 201 3624-3644AAGGGUTUAGACUGGAGCCAACA 560 3622-3644 AD-1136159.1 UUCCUGGCUGCUUCUAUCUUU202 3872-3892 AAAGAUAGAAGCAGCCAGGAAGA 561 3870-3892 AD-1136160.1UCCUGGCUGCUUCUAUCUUCU 203 3873-3893 AGAAGAUAGAAGCAGCCAGGAAG 5623871-3893 AD-1136161.1 CCUGGCUGCUUCUAUCUUCUU 204 3874-3894AAGAAGAUAGAAGCAGCCAGGAA 563 3872-3894 AD-1136162.1 CUGGCUGCUUCUAUCUUCUUU205 3875-3895 AAAGAAGAUAGAAGCAGCCAGGA 564 3873-3895 AD-1136163.1UGGCUGCUUCUAUCUUCUUUU 206 3876-3896 AAAAGAAGAUAGAAGCAGCCAGG 5653874-3896 AD-1136164.1 GCUGCUUCUAUCUUCUUUGCU 207 3878-3898AGCAAAGAAGAUAGAAGCAGCCA 566 3876-3898 AD-1136165.1 CUGCUUCUAUCUUCUUUGCCU208 3879-3899 AGGCAAAGAAGAUAGAAGCAGCC 567 3877-3899 AD-1136166.1UGCUUCUAUCUUCUUUGCCAU 209 3880-3900 AUGGCAAAGAAGAUAGAAGCAGC 5683878-3900 AD-1136167.1 GCUUCUAUCUUCUUUGCCAUU 210 3881-3901AAUGGCAAAGAAGAUAGAAGCAG 569 3879-3901 AD-1136168.1 CUUCUAUCUUCUUUGCCAUCU211 3882-3902 AGAUGGCAAAGAAGAUAGAAGCA 570 3880-3902 AD-1136169.1UUCUAUCUUCUUUGCCAUCAU 212 3883-3903 AUGATGGCAAAGAAGAUAGAAGC 5713881-3903 AD-1136170.1 UCUAUCUUCUUUGCCAUCAAU 213 3884-3904AUUGAUGGCAAAGAAGAUAGAAG 572 3882-3904 AD-1136171.1 CUAUCUUCUUUGCCAUCAAAU214 3885-3905 AUUUGATGGCAAAGAAGAUAGAA 573 3883-3905 AD-1136172.1UAUCUUCUUUGCCAUCAAAGU 215 3886-3906 ACUUUGAUGGCAAAGAAGAUAGA 5743884-3906 AD-1136173.1 AUCUUCUUUGCCAUCAAAGAU 216 3887-3907AUCUTUGAUGGCAAAGAAGAUAG 575 3885-3907 AD-1136174.1 CUUCUUUGCCAUCAAAGAUGU217 3889-3909 ACAUCUUUGAUGGCAAAGAAGAU 576 3887-3909 AD-1136175.1GAGCUCAGCACACAGGUAAUU 218 3924-3944 AAUUACCUGUGUGCUGAGCUCGA 5773922-3944 AD-1136176.1 CUCAGCACACAGGUAAUAACU 219 3927-3947AGUUAUUACCUGUGUGCUGAGCU 578 3925-3947 AD-1136177.1 UCAGCACACAGGUAAUAACGU220 3928-3948 ACGUUAUUACCUGUGUGCUGAGC 579 3926-3948 AD-1136178.1CAGCACACAGGUAAUAACGUU 221 3929-3949 AACGUUAUUACCUGUGUGCUGAG 5803927-3949 AD-1136179.1 ACAGGUAAUAACGUGAAGGAU 222 3935-3955AUCCTUCACGUUAUUACCUGUGU 581 3933-3955 AD-1136180.1 CAGGUAAUAACGUGAAGGAAU223 3936-3956 AUUCCUTCACGUUAUUACCUGUG 582 3934-3956 AD-1136181.1AGGUAAUAACGUGAAGGAACU 224 3937-3957 AGUUCCTUCACGUUAUUACCUGU 5833935-3957 AD-1136182.1 AUAACGUGAAGGAACUCUUCU 225 3942-3962AGAAGAGUUCCUUCACGUUAUUA 584 3940-3962 AD-1136183.1 UAACGUGAAGGAACUCUUCCU226 3943-3963 AGGAAGAGUUCCUUCACGUUAUU 585 3941-3963 AD-1136184.1CCCAGAAAACUGCAAACCCUU 227 4042-4062 AAGGGUTUGCAGUUUUCUGGGAC 5864040-4062 AD-1136185.1 CACAGAACAUGGAUCUAUUAU 228 4145-4165AUAATAGAUCCAUGUUCUGUGGU 587 4143-4165 AD-1136186.1 ACAGAACAUGGAUCUAUUAAU229 4146-4166 AUUAAUAGAUCCAUGUUCUGUGG 588 4144-4166 AD-1136187.1CAGAACAUGGAUCUAUUAAAU 230 4147-4167 AUUUAAUAGAUCCAUGUUCUGUG 5894145-4167 AD-1136188.1 AGAACAUGGAUCUAUUAAAGU 231 4148-4168ACUUUAAUAGAUCCAUGUUCUGU 590 4146-4168 AD-1136189.1 GAACAUGGAUCUAUUAAAGUU232 4149-4169 AACUUUAAUAGAUCCAUGUUCUG 591 4147-4169 AD-1136190.1AACAUGGAUCUAUUAAAGUCU 233 4150-4170 AGACUUUAAUAGAUCCAUGUUCU 5924148-4170 AD-1136191.1 ACAUGGAUCUAUUAAAGUCAU 234 4151-4171AUGACUTUAAUAGAUCCAUGUUC 593 4149-4171 AD-1136192.1 CAUGGAUCUAUUAAAGUCACU235 4152-4172 AGUGACTUUAAUAGAUCCAUGUU 594 4150-4172 AD-1136193.1AUGGAUCUAUUAAAGUCACAU 236 4153-4173 AUGUGACUUUAAUAGAUCCAUGU 5954151-4173 AD-1136194.1 UGGAUCUAUUAAAGUCACAGU 237 4154-4174ACUGTGACUUUAAUAGAUCCAUG 596 4152-4174 AD-1136195.1 GGAUCUAUUAAAGUCACAGAU238 4155-4175 AUCUGUGACUUUAAUAGAUCCAU 597 4153-4175 AD-1136196.1GAUCUAUUAAAGUCACAGAAU 239 4156-4176 AUUCTGTGACUUUAAUAGAUCCA 5984154-4176 AD-1136197.1 ACAAUGAUAAGCAAAUUCAAU 240 4261-4281AUUGAAUUUGCUUAUCAUUGUGU 599 4259-4281 AD-1136198.1 CAAUGAUAAGCAAAUUCAAAU241 4262-4282 AUUUGAAUUUGCUUAUCAUUGUG 600 4260-4282 AD-1136199.1AUGAUAAGCAAAUUCAAAACU 242 4264-4284 AGUUTUGAAUUUGCUUAUCAUUG 6014262-4284 AD-1136200.1 AUGCCUAAAUGGUGAAUAUGU 243 4288-4308ACAUAUUCACCAUUUAGGCAUAA 602 4286-4308 AD-1136201.1 UGCCUAAAUGGUGAAUAUGCU244 4289-4309 AGCAUAUUCACCAUUUAGGCAUA 603 4287-4309 AD-1136202.1GCCUAAAUGGUGAAUAUGCAU 245 4290-4310 AUGCAUAUUCACCAUUUAGGCAU 6044288-4310 AD-1136203.1 CCUAAAUGGUGAAUAUGCAAU 246 4291-4311AUUGCATAUUCACCAUUUAGGCA 605 4289-4311 AD-1136204.1 CUAAAUGGUGAAUAUGCAAUU247 4292-4312 AAUUGCAUAUUCACCAUUUAGGC 606 4290-4312 AD-1136205.1AAUGGUGAAUAUGCAAUUAGU 248 4295-4315 ACUAAUUGCAUAUUCACCAUUUA 6074293-4315 AD-1136206.1 CGGGAAGGGUUUGUGCUAUUU 249 4357-4377AAAUAGCACAAACCCUUCCCGAC 608 4355-4377 AD-1136207.1 GGGAAGGGUUUGUGCUAUUCU250 4358-4378 AGAATAGCACAAACCCUUCCCGA 609 4356-4378 AD-1136208.1GGAAGGGUUUGUGCUAUUCCU 251 4359-4379 AGGAAUAGCACAAACCCUUCCCG 6104357-4379 AD-1136209.1 GUAUAACCUCAAGUUCUGAUU 252 4397-4417AAUCAGAACUUGAGGUUAUACAG 611 4395-4417 AD-1136210.1 UAUAACCUCAAGUUCUGAUGU253 4398-4418 ACAUCAGAACUUGAGGUUAUACA 612 4396-4418 AD-1136211.1CCUCAAGUUCUGAUGGUGUCU 254 4403-4423 AGACACCAUCAGAACUUGAGGUU 6134401-4423 AD-1136212.1 CAAGUUCUGAUGGUGUCUGUU 255 4406-4426AACAGACACCAUCAGAACUUGAG 614 4404-4426 AD-1136213.1 CCACAAACCUCUAGAAGCUUU256 4442-4462 AAAGCUTCUAGAGGUUUGUGGGA 615 4440-4462 AD-1136214.1ACAAACCUCUAGAAGCUUAAU 257 4444-4464 AUUAAGCUUCUAGAGGUUUGUGG 6164442-4464 AD-1136215.1 AAACCUCUAGAAGCUUAAACU 258 4446-4466AGUUUAAGCUUCUAGAGGUUUGU 617 4444-4466 AD-1136216.1 AACCUCUAGAAGCUUAAACCU259 4447-4467 AGGUUUAAGCUUCUAGAGGUUUG 618 4445-4467 AD-1136217.1UGGCCUUCAAACCAAUGAACU 260 4504-4524 AGUUCAUUGGUUUGAAGGCCAGG 6194502-4524 AD-1136218.1 GGCCUUCAAACCAAUGAACAU 261 4505-4525AUGUTCAUUGGUUUGAAGGCCAG 620 4503-4525 AD-1136219.1 GCCUUCAAACCAAUGAACAGU262 4506-4526 ACUGUUCAUUGGUUUGAAGGCCA 621 4504-4526 AD-1136220.1UCAAACCAAUGAACAGCAAAU 263 4510-4530 AUUUGCTGUUCAUUGGUUUGAAG 6224508-4530 AD-1136221.1 GAACAGCAAAGCAUAACCUUU 264 4520-4540AAAGGUUAUGCUUUGCUGUUCAU 623 4518-4540 AD-1136222.1 AACAGCAAAGCAUAACCUUGU265 4521-4541 ACAAGGTUAUGCUUUGCUGUUCA 624 4519-4541 AD-1136223.1AGCAAAGCAUAACCUUGAAUU 266 4524-4544 AAUUCAAGGUUAUGCUUUGCUGU 6254522-4544 AD-1136224.1 GCAAAGCAUAACCUUGAAUCU 267 4525-4545AGAUTCAAGGUUAUGCUUUGCUG 626 4523-4545 AD-1136225.1 CAAAGCAUAACCUUGAAUCUU268 4526-4546 AAGATUCAAGGUUAUGCUUUGCU 627 4524-4546 AD-1136226.1GCAUAACCUUGAAUCUAUACU 269 4530-4550 AGUAUAGAUUCAAGGUUAUGCUU 6284528-4550 AD-1136227.1 CAUAACCUUGAAUCUAUACUU 270 4531-4551AAGUAUAGAUUCAAGGUUAUGCU 629 4529-4551 AD-1136228.1 AUAACCUUGAAUCUAUACUCU271 4532-4552 AGAGUAUAGAUUCAAGGUUAUGC 630 4530-4552 AD-1136229.1UAACCUUGAAUCUAUACUCAU 272 4533-4553 AUGAGUAUAGAUUCAAGGUUAUG 6314531-4553 AD-1136230.1 AACCUUGAAUCUAUACUCAAU 273 4534-4554AUUGAGTAUAGAUUCAAGGUUAU 632 4532-4554 AD-1136231.1 ACCUUGAAUCUAUACUCAAAU274 4535-4555 AUUUGAGUAUAGAUUCAAGGUUA 633 4533-4555 AD-1136232.1CCUUGAAUCUAUACUCAAAUU 275 4536-4556 AAUUTGAGUAUAGAUUCAAGGUU 6344534-4556 AD-1136233.1 AAUCUAUACUCAAAUUUUGCU 276 4541-4561AGCAAAAUUUGAGUAUAGAUUCA 635 4539-4561 AD-1136234.1 AUCUAUACUCAAAUUUUGCAU277 4542-4562 AUGCAAAAUUUGAGUAUAGAUUC 636 4540-4562 AD-1136235.1GGUUAAAUCCUCUAACCAUCU 278 4579-4599 AGAUGGTUAGAGGAUUUAACCUU 6374577-4599 AD-1136236.1 UAAAUCCUCUAACCAUCUUUU 279 4582-4602AAAAGATGGUUAGAGGAUUUAAC 638 4580-4602 AD-1136237.1 AAAUCCUCUAACCAUCUUUGU280 4583-4603 ACAAAGAUGGUUAGAGGAUUUAA 639 4581-4603 AD-1136238.1AAUCCUCUAACCAUCUUUGAU 281 4584-4604 AUCAAAGAUGGUUAGAGGAUUUA 6404582-4604 AD-1136239.1 AUCCUCUAACCAUCUUUGAAU 282 4585-4605AUUCAAAGAUGGUUAGAGGAUUU 641 4583-4605 AD-1136240.1 UCCUCUAACCAUCUUUGAAUU283 4586-4606 AAUUCAAAGAUGGUUAGAGGAUU 642 4584-4606 AD-1136241.1CCUCUAACCAUCUUUGAAUCU 284 4587-4607 AGAUTCAAAGAUGGUUAGAGGAU 6434585-4607 AD-1136242.1 CUCUAACCAUCUUUGAAUCAU 285 4588-4608AUGATUCAAAGAUGGUUAGAGGA 644 4586-4608 AD-1136243.1 UCUAACCAUCUUUGAAUCAUU286 4589-4609 AAUGAUUCAAAGAUGGUUAGAGG 645 4587-4609 AD-1136244.1CUAACCAUCUUUGAAUCAUUU 287 4590-4610 AAAUGATUCAAAGAUGGUUAGAG 6464588-4610 AD-1136245.1 UAACCAUCUUUGAAUCAUUGU 288 4591-4611ACAATGAUUCAAAGAUGGUUAGA 647 4589-4611 AD-1136246.1 AACCAUCUUUGAAUCAUUGGU289 4592-4612 ACCAAUGAUUCAAAGAUGGUUAG 648 4590-4612 AD-1136247.1CCAUCUUUGAAUCAUUGGAAU 290 4594-4614 AUUCCAAUGAUUCAAAGAUGGUU 6494592-4614 AD-1136248.1 CAUCUUUGAAUCAUUGGAAAU 291 4595-4615AUUUCCAAUGAUUCAAAGAUGGU 650 4593-4615 AD-1136249.1 AUCUUUGAAUCAUUGGAAAGU292 4596-4616 ACUUTCCAAUGAUUCAAAGAUGG 651 4594-4616 AD-1136250.1CUUUGAAUCAUUGGAAAGAAU 293 4598-4618 AUUCTUTCCAAUGAUUCAAAGAU 6524596-4618 AD-1136251.1 GAAAGAAUAAAGAAUGAAACU 294 4611-4631AGUUTCAUUCUUUAUUCUUUCCA 653 4609-4631 AD-1136252.1 AAAGAAUAAAGAAUGAAACAU295 4612-4632 AUGUTUCAUUCUUUAUUCUUUCC 654 4610-4632 AD-1136253.1GAAUGAAACAAAUUCAAGGUU 296 4622-4642 AACCUUGAAUUUGUUUCAUUCUU 6554620-4642 AD-1136254.1 AUGAAACAAAUUCAAGGUUAU 297 4624-4644AUAACCTUGAAUUUGUUUCAUUC 656 4622-4644 AD-1136255.1 AACAAAUUCAAGGUUAAUUGU298 4628-4648 ACAAUUAACCUUGAAUUUGUUUC 657 4626-4648 AD-1136256.1UGAAGCUGCAUAAAGCAAGAU 299 4660-4680 AUCUTGCUUUAUGCAGCUUCACA 6584658-4680 AD-1136257.1 AAGCUGCAUAAAGCAAGAUUU 300 4662-4682AAAUCUTGCUUUAUGCAGCUUCA 659 4660-4682 AD-1136258.1 AGCUGCAUAAAGCAAGAUUAU301 4663-4683 AUAATCTUGCUUUAUGCAGCUUC 660 4661-4683 AD-1136259.1GCUGCAUAAAGCAAGAUUACU 302 4664-4684 AGUAAUCUUGCUUUAUGCAGCUU 6614662-4684 AD-1136260.1 CUGCAUAAAGCAAGAUUACUU 303 4665-4685AAGUAAUCUUGCUUUAUGCAGCU 662 4663-4685 AD-1136261.1 UGCAUAAAGCAAGAUUACUCU304 4666-4686 AGAGUAAUCUUGCUUUAUGCAGC 663 4664-4686 AD-1136262.1CAUAAAGCAAGAUUACUCUAU 305 4668-4688 AUAGAGTAAUCUUGCUUUAUGCA 6644666-4688 AD-1136263.1 UAAAGCAAGAUUACUCUAUAU 306 4670-4690AUAUAGAGUAAUCUUGCUUUAUG 665 4668-4690 AD-1136264.1 AUACAAAAAUCCAACCAACUU307 4690-4710 AAGUTGGUUGGAUUUUUGUAUUA 666 4688-4710 AD-1136265.1UACAAAAAUCCAACCAACUCU 308 4691-4711 AGAGTUGGUUGGAUUUUUGUAUU 6674689-4711 AD-1136266.1 ACAAAAAUCCAACCAACUCAU 309 4692-4712AUGAGUTGGUUGGAUUUUUGUAU 668 4690-4712 AD-1136267.1 CAAAAAUCCAACCAACUCAAU310 4693-4713 AUUGAGTUGGUUGGAUUUUUGUA 669 4691-4713 AD-1136268.1AAAAAUCCAACCAACUCAAUU 311 4694-4714 AAUUGAGUUGGUUGGAUUUUUGU 6704692-4714 AD-1136269.1 AAAAUCCAACCAACUCAAUUU 312 4695-4715AAAUTGAGUUGGUUGGAUUUUUG 671 4693-4715 AD-1136270.1 AAAUCCAACCAACUCAAUUAU313 4696-4716 AUAATUGAGUUGGUUGGAUUUUU 672 4694-4716 AD-1136271.1AAUCCAACCAACUCAAUUAUU 314 4697-4717 AAUAAUUGAGUUGGUUGGAUUUU 6734695-4717 AD-1136272.1 AUCCAACCAACUCAAUUAUUU 315 4698-4718AAAUAAUUGAGUUGGUUGGAUUU 674 4696-4718 AD-1136273.1 UCCAACCAACUCAAUUAUUGU316 4699-4719 ACAAUAAUUGAGUUGGUUGGAUU 675 4697-4719 AD-1136274.1CCAACCAACUCAAUUAUUGAU 317 4700-4720 AUCAAUAAUUGAGUUGGUUGGAU 6764698-4720 AD-1136275.1 CAACCAACUCAAUUAUUGAGU 318 4701-4721ACUCAAUAAUUGAGUUGGUUGGA 677 4699-4721 AD-1136276.1 AACCAACUCAAUUAUUGAGCU319 4702-4722 AGCUCAAUAAUUGAGUUGGUUGG 678 4700-4722 AD-1136277.1ACCAACUCAAUUAUUGAGCAU 320 4703-4723 AUGCTCAAUAAUUGAGUUGGUUG 6794701-4723 AD-1136278.1 CCAACUCAAUUAUUGAGCACU 321 4704-4724AGUGCUCAAUAAUUGAGUUGGUU 680 4702-4724 AD-1136279.1 CGUACAAUGUUCUAGAUUUCU322 4723-4743 AGAAAUCUAGAACAUUGUACGUG 681 4721-4743 AD-1136280.1GUACAAUGUUCUAGAUUUCUU 323 4724-4744 AAGAAAUCUAGAACAUUGUACGU 6824722-4744 AD-1136281.1 UACAAUGUUCUAGAUUUCUUU 324 4725-4745AAAGAAAUCUAGAACAUUGUACG 683 4723-4745 AD-1136282.1 ACAAUGUUCUAGAUUUCUUUU325 4726-4746 AAAAGAAAUCUAGAACAUUGUAC 684 4724-4746 AD-1136283.1CAAUGUUCUAGAUUUCUUUCU 326 4727-4747 AGAAAGAAAUCUAGAACAUUGUA 6854725-4747 AD-1136284.1 AAUGUUCUAGAUUUCUUUCCU 327 4728-4748AGGAAAGAAAUCUAGAACAUUGU 686 4726-4748 AD-1136285.1 GUUCUAGAUUUCUUUCCCUUU328 4731-4751 AAAGGGAAAGAAAUCUAGAACAU 687 4729-4751 AD-1136286.1UUCUAGAUUUCUUUCCCUUCU 329 4732-4752 AGAAGGGAAAGAAAUCUAGAACA 6884730-4752 AD-1136287.1 UCUUUACAUUUGAAGCCAAAU 330 4817-4837AUUUGGCUUCAAAUGUAAAGAUU 689 4815-4837 AD-1136288.1 CUUUACAUUUGAAGCCAAAGU331 4818-4838 ACUUTGGCUUCAAAUGUAAAGAU 690 4816-4838 AD-1136289.1UUUACAUUUGAAGCCAAAGUU 332 4819-4839 AACUTUGGCUUCAAAUGUAAAGA 6914817-4839 AD-1136290.1 ACAUUUGAAGCCAAAGUAAUU 333 4822-4842AAUUACTUUGGCUUCAAAUGUAA 692 4820-4842 AD-1136291.1 AUUUGAAGCCAAAGUAAUUUU334 4824-4844 AAAAUUACUUUGGCUUCAAAUGU 693 4822-4844 AD-1136292.1GAAGCCAAAGUAAUUUCCACU 335 4828-4848 AGUGGAAAUUACUUUGGCUUCAA 6944826-4848 AD-1136293.1 AAGCCAAAGUAAUUUCCACCU 336 4829-4849AGGUGGAAAUUACUUUGGCUUCA 695 4827-4849 AD-1136294.1 CCAAAGUAAUUUCCACCUAGU337 4832-4852 ACUAGGTGGAAAUUACUUUGGCU 696 4830-4852 AD-1136295.1CAAAGUAAUUUCCACCUAGAU 338 4833-4853 AUCUAGGUGGAAAUUACUUUGGC 6974831-4853 AD-1136296.1 AAAGUAAUUUCCACCUAGAAU 339 4834-4854AUUCTAGGUGGAAAUUACUUUGG 698 4832-4854 AD-1136297.1 GCAAGAUCUUGUCUCUGAAGU340 5134-5154 ACUUCAGAGACAAGAUCUUGCUC 699 5132-5154 AD-1136298.1AGGUAGAAGAGCCAAGAAGCU 341 5201-5221 AGCUTCTUGGCUCUUCUACCUCU 7005199-5221 AD-1136299.1 CUAGCUCUGUCUCUUCUGUCU 342 5234-5254AGACAGAAGAGACAGAGCUAGGA 701 5232-5254 AD-1136300.1 UAGCUCUGUCUCUUCUGUCUU343 5235-5255 AAGACAGAAGAGACAGAGCUAGG 702 5233-5255 AD-1136301.1AGCUCUGUCUCUUCUGUCUCU 344 5236-5256 AGAGACAGAAGAGACAGAGCUAG 7035234-5256 AD-1136302.1 AUCUUUGUGAUCUUGGACUGU 345 5257-5277ACAGUCCAAGAUCACAAAGAUAG 704 5255-5277 AD-1136303.1 CUUCCUGUGAUCCAUUUUACU346 5286-5306 AGUAAAAUGGAUCACAGGAAGGG 705 5284-5306 AD-1136304.1UUCCUGUGAUCCAUUUUACUU 347 5287-5307 AAGUAAAAUGGAUCACAGGAAGG 7065285-5307 AD-1136305.1 UCCUGUGAUCCAUUUUACUGU 348 5288-5308ACAGUAAAAUGGAUCACAGGAAG 707 5286-5308 AD-1136306.1 CCUGUGAUCCAUUUUACUGCU349 5289-5309 AGCAGUAAAAUGGAUCACAGGAA 708 5287-5309 AD-1136307.1CUGUGAUCCAUUUUACUGCAU 350 5290-5310 AUGCAGTAAAAUGGAUCACAGGA 7095288-5310 AD-1136308.1 CUAUAAAUAUAUGACCUGAAU 351 5360-5380AUUCAGGUCAUAUAUUUAUAGGC 710 5358-5380 AD-1136309.1 UGACCUGAAAACUCCAGUUAU352 5371-5391 AUAACUGGAGUUUUCAGGUCAUA 711 5369-5391 AD-1136310.1GACCUGAAAACUCCAGUUACU 353 5372-5392 AGUAACTGGAGUUUUCAGGUCAU 7125370-5392 AD-1136311.1 AAGGAUCUGCAGCUAUCUAAU 354 5395-5415AUUAGATAGCUGCAGAUCCUUUA 713 5393-5415 AD-1136312.1 AGGAUCUGCAGCUAUCUAAGU355 5396-5416 ACUUAGAUAGCUGCAGAUCCUUU 714 5394-5416 AD-1136313.1GCUAUCUAAGGCUUGGUUUUU 356 5406-5426 AAAAACCAAGCCUUAGAUAGCUG 7155404-5426 AD-1136314.1 CUAUCUAAGGCUUGGUUUUCU 357 5407-5427AGAAAACCAAGCCUUAGAUAGCU 716 5405-5427 AD-1136315.1 AUCUAAGGCUUGGUUUUCUUU358 5409-5429 AAAGAAAACCAAGCCUUAGAUAG 717 5407-5429 AD-1136316.1UCUAAGGCUUGGUUUUCUUAU 359 5410-5430 AUAAGAAAACCAAGCCUUAGAUA 7185408-5430 AD-1136317.1 CUAAGGCUUGGUUUUCUUACU 360 5411-5431AGUAAGAAAACCAAGCCUUAGAU 719 5409-5431 AD-1136318.1 UAAGGCUUGGUUUUCUUACUU361 5412-5432 AAGUAAGAAAACCAAGCCUUAGA 720 5410-5432 AD-1136319.1GGCUUGGUUUUCUUACUGUCU 362 5415-5435 AGACAGTAAGAAAACCAAGCCUU 7215413-5435 AD-1136320.1 GCUUGGUUUUCUUACUGUCAU 363 5416-5436AUGACAGUAAGAAAACCAAGCCU 722 5414-5436 AD-1136321.1 CUUGGUUUUCUUACUGUCAUU364 5417-5437 AAUGACAGUAAGAAAACCAAGCC 723 5415-5437 AD-1136322.1UUGGUUUUCUUACUGUCAUAU 365 5418-5438 AUAUGACAGUAAGAAAACCAAGC 7245416-5438 AD-1136323.1 UGGUUUUCUUACUGUCAUAUU 366 5419-5439AAUATGACAGUAAGAAAACCAAG 725 5417-5439 AD-1136324.1 GGUUUUCUUACUGUCAUAUGU367 5420-5440 ACAUAUGACAGUAAGAAAACCAA 726 5418-5440 AD-1136325.1UUUUCUUACUGUCAUAUGAUU 368 5422-5442 AAUCAUAUGACAGUAAGAAAACC 7275420-5442 AD-1136326.1 UUUCUUACUGUCAUAUGAUAU 369 5423-5443AUAUCAUAUGACAGUAAGAAAAC 728 5421-5443 AD-1136327.1 UCUUACUGUCAUAUGAUACCU370 5425-5445 AGGUAUCAUAUGACAGUAAGAAA 729 5423-5445 AD-1136328.1CUUACUGUCAUAUGAUACCUU 371 5426-5446 AAGGUAUCAUAUGACAGUAAGAA 7305424-5446 AD-1136329.1 UCUGCGGUUGGUAAAGAGAAU 372 5481-5501AUUCTCTUUACCAACCGCAGAAA 731 5479-5501 AD-1136330.1 UGGUGAUUUAAUCCCUACAUU373 5537-5557 AAUGTAGGGAUUAAAUCACCAUU 732 5535-5557 AD-1136331.1GGUGAUUUAAUCCCUACAUGU 374 5538-5558 ACAUGUAGGGAUUAAAUCACCAU 7335536-5558 AD-1136332.1 UGUAUAAAUCCAACCUUCUGU 375 5597-5617ACAGAAGGUUGGAUUUAUACAGU 734 5595-5617 AD-1136333.1 GUGCUUGAUGUCACUAAUAAU376 5679-5699 AUUATUAGUGACAUCAAGCACCA 735 5677-5699 AD-1136334.1UGCUUGAUGUCACUAAUAAAU 377 5680-5700 AUUUAUUAGUGACAUCAAGCACC 7365678-5700 AD-1136335.1 GCUUGAUGUCACUAAUAAAUU 378 5681-5701AAUUUAUUAGUGACAUCAAGCAC 737 5679-5701 AD-1136336.1 AUGUCACUAAUAAAUGAAACU379 5686-5706 AGUUTCAUUUAUUAGUGACAUCA 738 5684-5706 AD-1136337.1UGUCACUAAUAAAUGAAACUU 380 5687-5707 AAGUTUCAUUUAUUAGUGACAUC 7395685-5707 AD-1136338.1 UAAUAAAUGAAACUGUCAGCU 381 5693-5713AGCUGACAGUUUCAUUUAUUAGU 740 5691-5713

TABLE 3Modified Sense and Antisense Strand Sequences of Xanthine Dehydrogenase dsRNA AgentsDuplex Name Sense Sequence 5′ to 3′ SEQ ID NO:Antisense Sequence 5′ to 3′ SEQ ID NO: mRNA Target Sequence SEQ ID NO:AD-1135979.1 usasccugCfcAfGfUfgucucuua 741asCfsuaaGfagacacuGfgCfagguasgs 1100 ACUACCUGCCAGUGUCUCUUA 1459 guL96 uGG AD-1135980.1 ascscugcCfaGfUfGfucucuuag 742asCfscuaAfgagacacUfgGfcaggusas 1101 CUACCUGCCAGUGUCUCUUAG 1460 guL96 gGA AD-1135981.1 cscsugccAfgUfGfUfcucuuagg 743asUfsccuAfagagacaCfuGfgcaggsus 1102 UACCUGCCAGUGUCUCUUAGG 1461 auL96 aAG AD-1135982.1 usgsccagUfgUfCfUfcuuaggag 744asAfscucCfuaagagaCfaCfuggcasgs 1103 CCUGCCAGUGUCUCUUAGGAG 1462 uuL96 gUG AD-1135983.1 gscscaguGfuCfUfCfuuaggagu 745asCfsacdTc(C2p)uaagagAfcAfcugg 1104 CUGCCAGUGUCUCUUAGGAGU 1463 guL96csasg GA AD-1135984.1 cscsagugUfcUfCfUfuaggagug 746asUfscadCu(C2p)cuaagaGfaCfacug 1105 UGCCAGUGUCUCUUAGGAGUG 1464 auL96gscsa AG AD-1135985.1 csasguguCfuCfUfUfaggaguga 747asCfsucaCfuccuaagAfgAfcacugsgs 1106 GCCAGUGUCUCUUAGGAGUGA 1465 guL96 cGG AD-1135986.1 asgsugucUfcUfUfAfggagugag 748asCfscucAfcuccuaaGfaGfacacusgs 1107 CCAGUGUCUCUUAGGAGUGAG 1466 guL96 gGU AD-1135987.1 usgsgagaAfaAfAfUfgcagaucc 749asUfsggdAu(C2p)ugcauuUfuUfcucc 1108 GGUGGAGAAAAAUGCAGAUCC 1467 auL96ascsc AG AD-1135988.1 cscsagagAfcAfAfCfccuuuugg 750asGfsccaAfaaggguuGfuCfucuggsas 1109 AUCCAGAGACAACCCUUUUGG 1468 cuL96 uCC AD-1135989.1 asgsagacAfaCfCfCfuuuuggcc 751asAfsggdCc(Agn)aaagggUfuGfucuc 1110 CCAGAGACAACCCUUUUGGCC 1469 uuL96usgsg UA AD-1135990.1 gscsacagUfgAfUfGfcucuccaa 752asCfsuudGg(Agn)gagcauCfaCfugug 1111 UUGCACAGUGAUGCUCUCCAA 1470 guL96csasa GU AD-1135991.1 csascaguGfaUfGfCfucuccaag 753asAfscudTg(G2p)agagcaUfcAfcugu 1112 UGCACAGUGAUGCUCUCCAAG 1471 uuL96gscsa UA AD-1135992.1 gsasugcuCfuCfCfAfaguaugau 754asGfsaucAfuacuuggAfgAfgcaucsas 1113 GUGAUGCUCUCCAAGUAUGAU 1472 cuL96 cCG AD-1135993.1 asusgcucUfcCfAfAfguaugauc 755asCfsgauCfauacuugGfaGfagcauscs 1114 UGAUGCUCUCCAAGUAUGAUC 1473 guL96 aGU AD-1135994.1 usgscucuCfcAfAfGfuaugaucg 756asAfscgaUfcauacuuGfgAfgagcasus 1115 GAUGCUCUCCAAGUAUGAUCG 1474 uuL96 cUC AD-1135995.1 gscsucucCfaAfGfUfaugaucgu 757asGfsacgAfucauacuUfgGfagagcsas 1116 AUGCUCUCCAAGUAUGAUCGU 1475 cuL96 uCU AD-1135996.1 csuscuccAfaGfUfAfugaucguc 758asAfsgacGfaucauacUfuGfgagagscs 1117 UGCUCUCCAAGUAUGAUCGUC 1476 uuL96 aUG AD-1135997.1 gsasucguCfuGfCfAfgaacaaga 759asAfsucdTu(G2p)uucugcAfgAfcgau 1118 AUGAUCGUCUGCAGAACAAGA 1477 uuL96csasu UC AD-1135998.1 csgsucugCfaGfAfAfcaagaucg 760asAfscgaUfcuuguucUfgCfagacgsas 1119 AUCGUCUGCAGAACAAGAUCG 1478 uuL96 uUC AD-1135999.1 gsuscugcAfgAfAfCfaagaucgu 761asGfsacgAfucuuguuCfuGfcagacsgs 1120 UCGUCUGCAGAACAAGAUCGU 1479 cuL96 aCC AD-1136000.1 uscsugcaGfaAfCfAfagaucguc 762asGfsgacGfaucuuguUfcUfgcagascs 1121 CGUCUGCAGAACAAGAUCGUC 1480 cuL96 gCA AD-1136001.1 gscsagaaCfaAfGfAfucguccac 763asAfsgudGg(Agn)cgaucuUfgUfucug 1122 CUGCAGAACAAGAUCGUCCAC 1481 uuL96csasg UU AD-1136002.1 csasgaacAfaGfAfUfcguccacu 764asAfsagdTg(G2p)acgaucUfuGfuucu 1123 UGCAGAACAAGAUCGUCCACU 1482 uuL96gscsa UU AD-1136003.1 gsasacaaGfaUfCfGfuccacuuu 765asAfsaadAg(Tgn)ggacgaUfcUfuguu 1124 CAGAACAAGAUCGUCCACUUU 1483 uuL96csusg UC AD-1136004.1 asascaagAfuCfGfUfccacuuuu 766asGfsaaaAfguggacgAfuCfuuguuscs 1125 AGAACAAGAUCGUCCACUUUU 1484 cuL96 ucu AD-1136005.1 ascsaagaUfcGfUfCfcacuuuuc 767asAfsgaaAfaguggacGfaUfcuugusus 1126 GAACAAGAUCGUCCACUUUUC 1485 uuL96 cUG AD-1136006.1 csasagauCfgUfCfCfacuuuucu 768asCfsagaAfaaguggaCfgAfucuugsus 1127 AACAAGAUCGUCCACUUUUCU 1486 guL96 uGC AD-1136007.1 asasgaucGfuCfCfAfcuuuucug 769asGfscagAfaaaguggAfcGfaucuusgs 1128 ACAAGAUCGUCCACUUUUCUG 1487 cuL96 uCC AD-1136008.1 csgsuccaCfuUfUfUfcugccaau 770asCfsaudTg(G2p)cagaaaAfgUfggac 1129 AUCGUCCACUUUUCUGCCAAU 1488 guL96gsasu GC AD-1136009.1 gsusccacUfuUfUfCfugccaaug 771asGfscadTu(G2p)gcagaaAfaGfugga 1130 UCGUCCACUUUUCUGCCAAUG 1489 cuL96csgsa CC AD-1136010.1 usgscuccUfuGfCfAfccauguug 772asGfscaaCfauggugcAfaGfgagcasgs 1131 UCUGCUCCUUGCACCAUGUUG 1490 cuL96 aCA AD-1136011.1 uscscuugCfaCfCfAfuguugcag 773asAfscudGc(Agn)acauggUfgCfaagg 1132 GCUCCUUGCACCAUGUUGCAG 1491 uuL96asgsc UG AD-1136012.1 ususgcacCfaUfGfUfugcaguga 774asGfsucdAc(Tgn)gcaacaUfgGfugca 1133 CCUUGCACCAUGUUGCAGUGA 1492 cuL96asgsg CA AD-1136013.1 csasccauGfuUfGfCfagugacaa 775asGfsuudGu(C2p)acugcaAfcAfuggu 1134 UGCACCAUGUUGCAGUGACAA 1493 cuL96gscsa CU AD-1136014.1 ascscaugUfuGfCfAfgugacaac 776asAfsgudTg(Tgn)cacugcAfaCfaugg 1135 GCACCAUGUUGCAGUGACAAC 1494 uuL96usgsc UG AD-1136015.1 asgsgagaGfaAfUfUfgccaaaag 777asGfscudTu(Tgn)ggcaauUfcUfcucc 1136 GCAGGAGAGAAUUGCCAAAAG 1495 cuL96usgsc CC AD-1136016.1 gsgsagagAfaUfUfGfccaaaagc 778asGfsgcdTu(Tgn)uggcaaUfuCfucuc 1137 CAGGAGAGAAUUGCCAAAAGC 1496 cuL96csusg CA AD-1136017.1 usgsgcauCfgUfCfAfugaguaug 779asAfscauAfcucaugaCfgAfugccasgs 1138 CCUGGCAUCGUCAUGAGUAUG 1497 uuL96 gUA AD-1136018.1 gsgscaucGfuCfAfUfgaguaugu 780asUfsacdAu(Agn)cucaugAfcGfaugc 1139 CUGGCAUCGUCAUGAGUAUGU 1498 auL96csasg AC AD-1136019.1 gscsaucgUfcAfUfGfaguaugua 781asGfsuacAfuacucauGfaCfgaugcscs 1140 UGGCAUCGUCAUGAGUAUGUA 1499 cuL96 aCA AD-1136020.1 csasucguCfaUfGfAfguauguac 782asUfsgudAc(Agn)uacucaUfgAfcgau 1141 GGCAUCGUCAUGAGUAUGUAC 1500 auL96gscsc AC AD-1136021.1 asuscgucAfuGfAfGfuauguaca 783asGfsugdTa(C2p)auacucAfuGfacga 1142 GCAUCGUCAUGAGUAUGUACA 1501 cuL96usgsc CA AD-1136022.1 csgsucauGfaGfUfAfuguacaca 784asGfsugdTg(Tgn)acauacUfcAfugac 1143 AUCGUCAUGAGUAUGUACACA 1502 cuL96gsasu CU AD-1136023.1 gsuscaugAfgUfAfUfguacacac 785asAfsgudGu(G2p)uacauaCfuCfauga 1144 UCGUCAUGAGUAUGUACACAC 1503 uuL96csgsa UG AD-1136024.1 uscsaugaGfuAfUfGfuacacacu 786asCfsagdTg(Tgn)guacauAfcUfcaug 1145 CGUCAUGAGUAUGUACACACU 1504 guL96ascsg GC AD-1136025.1 asasugccUfuCfCfAfaggaaauc 787asAfsgadTu(Tgn)ccuuggAfaGfgcau 1146 AGAAUGCCUUCCAAGGAAAUC 1505 uuL96uscsu UG AD-1136026.1 asusgccuUfcCfAfAfggaaaucu 788asCfsagaUfuuccuugGfaAfggcausus 1147 GAAUGCCUUCCAAGGAAAUCU 1506 guL96 cGU AD-1136027.1 usgsccuuCfcAfAfGfgaaaucug 789asAfscagAfuuuccuuGfgAfaggcasus 1148 AAUGCCUUCCAAGGAAAUCUG 1507 uuL96 uUG AD-1136028.1 gscscuucCfaAfGfGfaaaucugu 790asCfsacaGfauuuccuUfgGfaaggcsas 1149 AUGCCUUCCAAGGAAAUCUGU 1508 guL96 uGC AD-1136029.1 cscsuuccAfaGfGfAfaaucugug 791asGfscacAfgauuuccUfuGfgaaggscs 1150 UGCCUUCCAAGGAAAUCUGUG 1509 cuL96 aCC AD-1136030.1 gsasgaugAfaGfUfUfcaagaaua 792asAfsuadTu(C2p)uugaacUfuCfaucu 1151 UUGAGAUGAAGUUCAAGAAUA 1510 uuL96csasa UG AD-1136031.1 gsasugaaGfuUfCfAfagaauaug 793asGfscauAfuucuugaAfcUfucaucsus 1152 GAGAUGAAGUUCAAGAAUAUG 1511 cuL96 cCU AD-1136032.1 asasguucAfaGfAfAfuaugcugu 794asAfsacdAg(C2p)auauucUfuGfaacu 1153 UGAAGUUCAAGAAUAUGCUGU 1512 uuL96uscsa UU AD-1136033.1 asgsuucaAfgAfAfUfaugcuguu 795asAfsaadCa(G2p)cauauuCfuUfgaac 1154 GAAGUUCAAGAAUAUGCUGUU 1513 uuL96USUSC UC AD-1136034.1 gsusucaaGfaAfUfAfugcuguuu 796asGfsaadAc(Agn)gcauauUfcUfugaa 1155 AAGUUCAAGAAUAUGCUGUUU 1514 cuL96csusu CC AD-1136035.1 ususcaagAfaUfAfUfgcuguuuc 797asGfsgaaAfcagcauaUfuCfuugaascs 1156 AGUUCAAGAAUAUGCUGUUUC 1515 cuL96 uCU AD-1136036.1 asasgaauAfuGfCfUfguuuccua 798asAfsuadGg(Agn)aacagcAfuAfuucu 1157 UCAAGAAUAUGCUGUUUCCUA 1516 uuL96usgsa UG AD-1136037.1 gsgsgaguAfuUfUfCfucagcauu 799asGfsaadTg(C2p)ugagaaAfuAfcucc 1158 GGGGGAGUAUUUCUCAGCAUU 1517 cuL96CSCSC CA AD-1136038.1 gsgsaguaUfuUfCfUfcagcauuc 800asUfsgadAu(G2p)cugagaAfaUfacuC 1159 GGGGAGUAUUUCUCAGCAUUC 1518 auL96CSCSC AA AD-1136039.1 gsasguauUfuCfUfCfagcauuca 801asUfsugaAfugcugagAfaAfuacucscs 1160 GGGAGUAUUUCUCAGCAUUCA 1519 auL96 cAG AD-1136040.1 asgsuauuUfcUfCfAfgcauucaa 802asCfsuugAfaugcugaGfaAfauacuscs 1161 GGAGUAUUUCUCAGCAUUCAA 1520 guL96 cGC AD-1136041.1 gsusauuuCfuCfAfGfcauucaag 803asGfscudTg(Agn)augcugAfgAfaaua 1162 GAGUAUUUCUCAGCAUUCAAG 1521 cuL96csusc CA AD-1136042.1 gsusggcaUfgAfGfAfguuuuauu 804asGfsaauAfaaacucuCfaUfgccacsus 1163 CAGUGGCAUGAGAGUUUUAUU 1522 cuL96 gCA AD-1136043.1 gsgscaugAfgAfGfUfuuuauuca 805asUfsugaAfuaaaacuCfuCfaugccsas 1164 GUGGCAUGAGAGUUUUAUUCA 1523 auL96 cAG AD-1136044.1 csasugagAfgUfUfUfuauucaag 806asGfscudTg(Agn)auaaaaCfuCfucau 1165 GGCAUGAGAGUUUUAUUCAAG 1524 cuL96gscsc CC AD-1136045.1 cscsucacCfcUfCfAfgcuucuuc 807asAfsgaaGfaagcugaGfgGfugaggsgs 1166 ACCCUCACCCUCAGCUUCUUC 1525 uuL96 uUU AD-1136046.1 uscsacccUfcAfGfCfuucuucuu 808asGfsaadGa(Agn)gaagcuGfaGfggug 1167 CCUCACCCUCAGCUUCUUCUU 1526 cuL96asgsg CA AD-1136047.1 uscsuucaAfgUfUfCfuaccugac 809asUfsgudCa(G2p)guagaaCfuUfgaag 1168 CUUCUUCAAGUUCUACCUGAC 1527 auL96asasg AG AD-1136048.1 ususucgcCfaGfUfGfcaacuuua 810asGfsuaaAfguugcacUfgGfcgaaasgs 1169 ACUUUCGCCAGUGCAACUUUA 1528 cuL96 uCU AD-1136049.1 ususcgccAfgUfGfCfaacuuuac 811asAfsguaAfaguugcaCfuGfgcgaasas 1170 CUUUCGCCAGUGCAACUUUAC 1529 uuL96 gUG AD-1136050.1 ususccagGfgUfUfUfguuuguuu 812asGfsaaaCfaaacaaaCfcCfuggaascs 1171 GGUUCCAGGGUUUGUUUGUUU 1530 cuL96 cCA AD-1136051.1 csasggguUfuGfUfUfuguuucau 813asAfsaugAfaacaaacAfaAfcccugsgs 1172 UCCAGGGUUUGUUUGUUUCAU 1531 uuL96 aUU AD-1136052.1 gsgsguuuGfuUfUfGfuuucauuu 814asGfsaaaUfgaaacaaAfcAfaacccsus 1173 CAGGGUUUGUUUGUUUCAUUU 1532 cuL96 gCC AD-1136053.1 gsgsuuugUfuUfGfUfuucauuuc 815asGfsgaaAfugaaacaAfaCfaaaccscs 1174 AGGGUUUGUUUGUUUCAUUUC 1533 cuL96 uCG AD-1136054.1 ususguuuGfuUfUfCfauuuccgc 816asAfsgcgGfaaaugaaAfcAfaacaasas 1175 GUUUGUUUGUUUCAUUUCCGC 1534 uuL96 cUG AD-1136055.1 ususccugGfgAfGfUfaacauaac 817asAfsguuAfuguuacuCfcCfaggaascs 1176 UGUUCCUGGGAGUAACAUAAC 1535 uuL96 aUG AD-1136056.1 gsgsgaguAfaCfAfUfaacuggaa 818asAfsuudCc(Agn)guuaugUfuAfcucc 1177 CUGGGAGUAACAUAACUGGAA 1536 uuL96csasg UU AD-1136057.1 usasacauAfaCfUfGfgaauuugu 819asUfsacaAfauuccagUfuAfuguuascs 1178 AGUAACAUAACUGGAAUUUGU 1537 auL96 uAA AD-1136058.1 asascauaAfcUfGfGfaauuugua 820asUfsuacAfaauuccaGfuUfauguusas 1179 GUAACAUAACUGGAAUUUGUA 1538 auL96 cAU AD-1136059.1 csgsaaggAfuAfAfGfguuacuug 821asAfscaaGfuaaccuuAfuCfcuucgscs 1180 UGCGAAGGAUAAGGUUACUUG 1539 uuL96 aUG AD-1136060.1 gsasaggaUfaAfGfGfuuacuugu 822asCfsacaAfguaaccuUfaUfccuucsgs 1181 GCGAAGGAUAAGGUUACUUGU 1540 guL96 cGU AD-1136061.1 gsgsgugaAfaAfUfCfaccuauga 823asUfsucaUfaggugauUfuUfcacccscs 1182 AGGGGUGAAAAUCACCUAUGA 1541 auL96 uAG AD-1136062.1 asasucacCfuAfUfGfaagaacua 824asGfsuadGu(Tgn)cuucauAfgGfugau 1183 AAAAUCACCUAUGAAGAACUA 1542 cuL96ususu CC AD-1136063.1 asuscaccUfaUfGfAfagaacuac 825asGfsgudAg(Tgn)ucuucaUfaGfguga 1184 AAAUCACCUAUGAAGAACUAC 1543 cuL96ususu CA AD-1136064.1 usasccagCfcAfUfUfaucacaau 826asAfsaudTg(Tgn)gauaauGfgCfuggu 1185 ACUACCAGCCAUUAUCACAAU 1544 uuL96asgsu UG AD-1136065.1 gsasacaaCfuCfCfUfuuuaugga 827asGfsuccAfuaaaaggAfgUfuguucsus 1186 AAGAACAACUCCUUUUAUGGA 1545 cuL96 uCC AD-1136066.1 gsgsccaaGfaGfCfAfcuucuacc 828asAfsggdTa(G2p)aagugcUfcUfuggc 1187 GUGGCCAAGAGCACUUCUACC 1546 uuL96csasc UG AD-1136067.1 gsasgcacUfuCfUfAfccuggaga 829asGfsucdTc(C2p)agguagAfaGfugcu 1188 AAGAGCACUUCUACCUGGAGA 1547 cuL96csusu CU AD-1136068.1 gscsacuuCfuAfCfCfuggagacu 830asGfsagdTc(Tgn)ccagguAfgAfagug 1189 GAGCACUUCUACCUGGAGACU 1548 cuL96csusc CA AD-1136069.1 csascuucUfaCfCfUfggagacuc 831asUfsgadGu(C2p)uccaggUfaGfaagu 1190 AGCACUUCUACCUGGAGACUC 1549 auL96gscsu AC AD-1136070.1 uscsacugCfaCfCfAfuugcuguu 832asGfsaadCa(G2p)caauggUfgCfagug 1191 ACUCACUGCACCAUUGCUGUU 1550 cuL96asgsu CC AD-1136071.1 csascugcAfcCfAfUfugcuguuc 833asGfsgadAc(Agn)gcaaugGfuGfcagu 1192 CUCACUGCACCAUUGCUGUUC 1551 cuL96gsasg CA AD-1136072.1 cscsauugCfuGfUfUfccaaaagg 834asGfsccuUfuuggaacAfgCfaauggsus 1193 CACCAUUGCUGUUCCAAAAGG 1552 cuL96 gCG AD-1136073.1 asgscucuUfuGfUfGfucuacaca 835asCfsugdTg(Tgn)agacacAfaAfgagc 1194 GGAGCUCUUUGUGUCUACACA 1553 guL96uscsc GA AD-1136074.1 gscsucuuUfgUfGfUfcuacacag 836asUfscudGu(G2p)uagacaCfaAfagag 1195 GAGCUCUUUGUGUCUACACAG 1554 auL96csusc AA AD-1136075.1 csuscuuuGfuGfUfCfuacacaga 837asUfsucdTg(Tgn)guagacAfcAfaaga 1196 AGCUCUUUGUGUCUACACAGA 1555 auL96gscsu AC AD-1136076.1 uscsuuugUfgUfCfUfacacagaa 838asGfsuudCu(G2p)uguagaCfaCfaaag 1197 GCUCUUUGUGUCUACACAGAA 1556 cuL96asgsc CA AD-1136077.1 csusuuguGfuCfUfAfcacagaac 839asUfsgudTc(Tgn)guguagAfcAfcaaa 1198 CUCUUUGUGUCUACACAGAAC 1557 auL96gsasg AC AD-1136078.1 ususugugUfcUfAfCfacagaaca 840asGfsugdTu(C2p)uguguaGfaCfacaa 1199 UCUUUGUGUCUACACAGAACA 1558 cuL96asgsa CC AD-1136079.1 ascsacagAfaCfAfCfcaugaaga 841asGfsucdTu(C2p)auggugUfuCfugug 1200 CUACACAGAACACCAUGAAGA 1559 cuL96usasg CC AD-1136080.1 gsasgcuuUfgUfUfGfcaaaaaug 842asAfscauUfuuugcaaCfaAfagcucsus 1201 CAGAGCUUUGUUGCAAAAAUG 1560 uuL96 gUU AD-1136081.1 asgscuuuGfuUfGfCfaaaaaugu 843asAfsacaUfuuuugcaAfcAfaagcuscs 1202 AGAGCUUUGUUGCAAAAAUGU 1561 uuL96 uUG AD-1136082.1 asgsgaucUfcUfCfUfcagaguau 844asAfsaudAc(Tgn)cugagaGfaGfaucc 1203 CCAGGAUCUCUCUCAGAGUAU 1562 uuL96usgsg UA AD-1136083.1 gsgsaucuCfuCfUfCfagaguauu 845asUfsaadTa(C2p)ucugagAfgAfgauc 1204 CAGGAUCUCUCUCAGAGUAUU 1563 auL96csusg AU AD-1136084.1 gsasucucUfcUfCfAfgaguauua 846asAfsuaaUfacucugaGfaGfagaucscs 1205 AGGAUCUCUCUCAGAGUAUUA 1564 uuL96 uUG AD-1136085.1 asuscucuCfuCfAfGfaguauuau 847asCfsauaAfuacucugAfgAfgagauscs 1206 GGAUCUCUCUCAGAGUAUUAU 1565 guL96 cGG AD-1136086.1 uscsucucUfcAfGfAfguauuaug 848asCfscauAfauacucuGfaGfagagasus 1207 GAUCUCUCUCAGAGUAUUAUG 1566 guL96 cGA AD-1136087.1 csuscucuCfaGfAfGfuauuaugg 849asUfsccaUfaauacucUfgAfgagagsas 1208 AUCUCUCUCAGAGUAUUAUGG 1567 auL96 uAA AD-1136088.1 uscsucucAfgAfGfUfauuaugga 850asUfsuccAfuaauacuCfuGfagagasgs 1209 UCUCUCUCAGAGUAUUAUGGA 1568 auL96 aAC AD-1136089.1 csuscucaGfaGfUfAfuuauggaa 851asGfsuudCc(Agn)uaauacUfcUfgaga 1210 CUCUCUCAGAGUAUUAUGGAA 1569 cuL96gsasg CG AD-1136090.1 uscsucagAfgUfAfUfuauggaac 852asCfsguuCfcauaauaCfuCfugagasgs 1211 UCUCUCAGAGUAUUAUGGAAC 1570 guL96 aGA AD-1136091.1 uscsagagUfaUfUfAfuggaacga 853asCfsucgUfuccauaaUfaCfucugasgs 1212 UCUCAGAGUAUUAUGGAACGA 1571 guL96 aGC AD-1136092.1 csasgaguAfuUfAfUfggaacgag 854asGfscucGfuuccauaAfuAfcucugsas 1213 CUCAGAGUAUUAUGGAACGAG 1572 cuL96 gCU AD-1136093.1 gscsugugCfaAfAfAfccaaccuu 855asGfsaadGg(Tgn)ugguuuUfgCfacag 1214 CGGCUGUGCAAAACCAACCUU 1573 cuL96cscsg CC AD-1136094.1 csusgugcAfaAfAfCfcaaccuuc 856asGfsgadAg(G2p)uugguuUfuGfcaca 1215 GGCUGUGCAAAACCAACCUUC 1574 cuL96gscsc CC AD-1136095.1 cscsugacAfcAfCfUfucaaccag 857asUfscudGg(Tgn)ugaaguGfuGfucag 1216 GACCUGACACACUUCAACCAG 1575 auL96gsusc AA AD-1136096.1 usgsacacAfcUfUfCfaaccagaa 858asCfsuudCu(G2p)guugaaGfuGfuguc 1217 CCUGACACACUUCAACCAGAA 1576 guL96asgsg GC AD-1136097.1 gsascacaCfuUfCfAfaccagaag 859asGfscudTc(Tgn)gguugaAfgUfgugu 1218 CUGACACACUUCAACCAGAAG 1577 cuL96csasg cu AD-1136098.1 ascsacuuCfaAfCfCfagaagcuu 860asCfsaagCfuucugguUfgAfagugusgs 1219 ACACACUUCAACCAGAAGCUU 1578 guL96 uGA AD-1136099.1 asusgccuAfgCfAfAfgcucucag 861asAfscudGa(G2p)agcuugCfuAfggca 1220 GAAUGCCUAGCAAGCUCUCAG 1579 uuL96ususc UA AD-1136100.1 cscsuagcAfaGfCfUfcucaguau 862asGfsaudAc(Tgn)gagagcUfuGfcuag 1221 UGCCUAGCAAGCUCUCAGUAU 1580 cuL96gscsa CA AD-1136101.1 csusagcaAfgCfUfCfucaguauc 863asUfsgadTa(C2p)ugagagCfuUfgcua 1222 GCCUAGCAAGCUCUCAGUAUC 1581 auL96gsgsc AU AD-1136102.1 usasgcaaGfcUfCfUfcaguauca 864asAfsugaUfacugagaGfcUfugcuasgs 1223 CCUAGCAAGCUCUCAGUAUCA 1582 uuL96 gUG AD-1136103.1 asgscaagCfuCfUfCfaguaucau 865asCfsaugAfuacugagAfgCfuugcusas 1224 CUAGCAAGCUCUCAGUAUCAU 1583 guL96 gGC AD-1136104.1 csasagcuCfuCfAfGfuaucaugc 866asAfsgcdAu(G2p)auacugAfgAfgcuu 1225 AGCAAGCUCUCAGUAUCAUGC 1584 uuL96gscsu UC AD-1136105.1 csuscggaAfgAfGfUfgagguuga 867asGfsucdAa(C2p)cucacuCfuUfccga 1226 UGCUCGGAAGAGUGAGGUUGA 1585 cuL96gscsa CA AD-1136106.1 asasggagAfaUfUfGfuuggaaaa 868asUfsuudTu(C2p)caacaaUfuCfuccu 1227 ACAAGGAGAAUUGUUGGAAAA 1586 auL96usgsu AG AD-1136107.1 csasccaaGfuUfUfGfgaauaagc 869asAfsgcuUfauuccaaAfcUfuggugsgs 1228 CCCACCAAGUUUGGAAUAAGC 1587 uuL96 gUU AD-1136108.1 ascscaagUfuUfGfGfaauaagcu 870asAfsagcUfuauuccaAfaCfuuggusgs 1229 CCACCAAGUUUGGAAUAAGCU 1588 uuL96 gUU AD-1136109.1 asgsuuccUfuUfUfCfugaaucag 871asCfscugAfuucagaaAfaGfgaacusgs 1230 ACAGUUCCUUUUCUGAAUCAG 1589 guL96 uGC AD-1136110.1 gsusuccuUfuUfCfUfgaaucagg 872asGfsccdTg(Agn)uucagaAfaAfggaa 1231 CAGUUCCUUUUCUGAAUCAGG 1590 cuL96csusg CA AD-1136111.1 ususccuuUfuCfUfGfaaucaggc 873asUfsgcdCu(G2p)auucagAfaAfagga 1232 AGUUCCUUUUCUGAAUCAGGC 1591 auL96ascsu AG AD-1136112.1 usascuucAfuGfUfGfuacacaga 874asAfsucdTg(Tgn)guacacAfuGfaagu 1233 CCUACUUCAUGUGUACACAGA 1592 uuL96asgsg UG AD-1136114.1 csasaggcCfuUfCfAfuaccaaaa 875asAfsuudTu(G2p)guaugaAfgGfccuu 1234 GCCAAGGCCUUCAUACCAAAA 1593 uuL96gsgsc UG AD-1136115.1 asasggccUfuCfAfUfaccaaaau 876asCfsauuUfugguaugAfaGfgccuusgs 1235 CCAAGGCCUUCAUACCAAAAU 1594 guL96 gGG AD-1136116.1 gscscuucAfuAfCfCfaaaauggu 877asGfsaccAfuuuugguAfuGfaaggcscs 1236 AGGCCUUCAUACCAAAAUGGU 1595 cuL96 uCC AD-1136117.1 csasccucUfaAfGfAfuuuauauc 878asUfsgauAfuaaaucuUfaGfaggugsgs 1237 CCCACCUCUAAGAUUUAUAUC 1596 auL96 gAG AD-1136118.1 ascscucuAfaGfAfUfuuauauca 879asCfsugaUfauaaaucUfuAfgaggusgs 1238 CCACCUCUAAGAUUUAUAUCA 1597 guL96 gGC AD-1136119.1 gscsgagaCfaAfGfCfacuaacac 880asAfsgudGu(Tgn)agugcuUfgUfcucg 1239 CAGCGAGACAAGCACUAACAC 1598 uuL96csusg UG AD-1136120.1 csgsagacAfaGfCfAfcuaacacu 881asCfsagdTg(Tgn)uagugcUfuGfucuc 1240 AGCGAGACAAGCACUAACACU 1599 guL96gscsu GU AD-1136121.1 gsasgacaAfgCfAfCfuaacacug 882asAfscadGu(G2p)uuagugCfuUfgucu 1241 GCGAGACAAGCACUAACACUG 1600 uuL96csgsc UG AD-1136122.1 asgsacaaGfcAfCfUfaacacugu 883asCfsacdAg(Tgn)guuaguGfcUfuguc 1242 CGAGACAAGCACUAACACUGU 1601 guL96uscsg GC AD-1136123.1 csgsgcuuGfuCfAfGfaccaucuu 884asCfsaagAfuggucugAfcAfagccgscs 1243 UGCGGCUUGUCAGACCAUCUU 1602 guL96 aGA AD-1136124.1 gsgscuugUfcAfGfAfccaucuug 885asUfscaaGfauggucuGfaCfaagccsgs 1244 GCGGCUUGUCAGACCAUCUUG 1603 auL96 cAA AD-1136125.1 gscsuuguCfaGfAfCfcaucuuga 886asUfsucdAa(G2p)auggucUfgAfcaag 1245 CGGCUUGUCAGACCAUCUUGA 1604 auL96CSCSg AA AD-1136126.1 csusugucAfgAfCfCfaucuugaa 887asUfsuudCa(Agn)gaugguCfuGfacaa 1246 GGCUUGUCAGACCAUCUUGAA 1605 auL96gscsc AA AD-1136127.1 ususgucaGfaCfCfAfucuugaaa 888asUfsuudTc(Agn)agauggUfcUfgaca 1247 GCUUGUCAGACCAUCUUGAAA 1606 auL96asgsc AG AD-1136128.1 usgsucagAfcCfAfUfcuugaaaa 889asCfsuuuUfcaagaugGfuCfugacasas 1248 CUUGUCAGACCAUCUUGAAAA 1607 guL96 gGG AD-1136129.1 gsuscagaCfcAfUfCfuugaaaag 890asCfscuuUfucaagauGfgUfcugacsas 1249 UUGUCAGACCAUCUUGAAAAG 1608 guL96 aGC AD-1136130.1 uscsagacCfaUfCfUfugaaaagg 891asGfsccuUfuucaagaUfgGfucugascs 1250 UGUCAGACCAUCUUGAAAAGG 1609 cuL96 aCU AD-1136131.1 ascsccuaCfaAfGfAfagaagaau 892asGfsaudTc(Tgn)ucuucuUfgUfaggg 1251 GAACCCUACAAGAAGAAGAAU 1610 cuL96ususc CC AD-1136132.1 csusgccaCfuGfGfGfuuuuauag 893asUfscuaUfaaaacccAfgUfggcagsas 1252 GUCUGCCACUGGGUUUUAUAG 1611 auL96 cAA AD-1136133.1 usgsccacUfgGfGfUfuuuauaga 894asUfsucuAfuaaaaccCfaGfuggcasgs 1253 UCUGCCACUGGGUUUUAUAGA 1612 auL96 aAC AD-1136134.1 csascuggGfuUfUfUfauagaaca 895asGfsugdTu(C2p)uauaaaAfcCfcagu 1254 GCCACUGGGUUUUAUAGAACA 1613 cuL96gsgsc CC AD-1136135.1 ascsugggUfuUfUfAfuagaacac 896asGfsgudGu(Tgn)cuauaaAfaCfccag 1255 CCACUGGGUUUUAUAGAACAC 1614 cuL96usgsg CC AD-1136136.1 gsgsguuuUfaUfAfGfaacaccca 897asUfsugdGg(Tgn)guucuaUfaAfaacc 1256 CUGGGUUUUAUAGAACACCCA 1615 auL96csasg AU AD-1136137.1 gsgsuuuuAfuAfGfAfacacccaa 898asAfsuudGg(G2p)uguucuAfuAfaaac 1257 UGGGUUUUAUAGAACACCCAA 1616 uuL96cscsa UC AD-1136138.1 usgsggcuAfcAfGfCfuuugagac 899asAfsgudCu(C2p)aaagcuGfuAfgccc 1258 UCUGGGCUACAGCUUUGAGAC 1617 uuL96asgsa UA AD-1136139.1 gsgsgcuaCfaGfCfUfuugagacu 900asUfsagdTc(Tgn)caaagcUfgUfagcc 1259 CUGGGCUACAGCUUUGAGACU 1618 auL96csasg AA AD-1136140.1 gscsuacaGfcUfUfUfgagacuaa 901asGfsuudAg(Tgn)cucaaaGfcUfguag 1260 GGGCUACAGCUUUGAGACUAA 1619 cuL96cscsc CU AD-1136141.1 csusacagCfuUfUfGfagacuaac 902asAfsgudTa(G2p)ucucaaAfgCfugua 1261 GGCUACAGCUUUGAGACUAAC 1620 uuL96gscsc UC AD-1136142.1 usascagcUfuUfGfAfgacuaacu 903asGfsagdTu(Agn)gucucaAfaGfcugu 1262 GCUACAGCUUUGAGACUAACU 1621 cuL96asgsc CA AD-1136143.1 ascsagcuUfuGfAfGfacuaacuc 904asUfsgadGu(Tgn)agucucAfaAfgcug 1263 CUACAGCUUUGAGACUAACUC 1622 auL96usasg AG AD-1136144.1 csasgcuuUfgAfGfAfcuaacuca 905asCfsugaGfuuagucuCfaAfagcugsus 1264 UACAGCUUUGAGACUAACUCA 1623 guL96 aGG AD-1136145.1 asgscuuuGfaGfAfCfuaacucag 906asCfscudGa(G2p)uuagucUfcAfaagc 1265 ACAGCUUUGAGACUAACUCAG 1624 guL96usgsu GG AD-1136146.1 csusuccaCfuAfCfUfucagcuau 907asCfsaudAg(C2p)ugaaguAfgUfggaa 1266 CCCUUCCACUACUUCAGCUAU 1625 guL96gsgsg GG AD-1136147.1 ususccacUfaCfUfUfcagcuaug 908asCfscauAfgcugaagUfaGfuggaasgs 1267 CCUUCCACUACUUCAGCUAUG 1626 guL96 gGG AD-1136148.1 gsusggcuUfgCfUfCfugaaguag 909asUfscudAc(Tgn)ucagagCfaAfgcca 1268 GGGUGGCUUGCUCUGAAGUAG 1627 auL96cscsc AA AD-1136149.1 usgsgcuuGfcUfCfUfgaaguaga 910asUfsucdTa(C2p)uucagaGfcAfagcc 1269 GGUGGCUUGCUCUGAAGUAGA 1628 auL96ascsc AA AD-1136150.1 gsgscuugCfuCfUfGfaaguagaa 911asUfsuudCu(Agn)cuucagAfgCfaagc 1270 GUGGCUUGCUCUGAAGUAGAA 1629 auL96csasc AU AD-1136151.1 gscsuugcUfcUfGfAfaguagaaa 912asAfsuudTc(Tgn)acuucaGfaGfcaag 1271 UGGCUUGCUCUGAAGUAGAAA 1630 uuL96cscsa UC AD-1136152.1 cscsuaacAfgGfAfGfaucauaag 913asUfscuuAfugaucucCfuGfuuaggscs 1272 UGCCUAACAGGAGAUCAUAAG 1631 auL96 aAA AD-1136153.1 csusaacaGfgAfGfAfucauaaga 914asUfsucdTu(Agn)ugaucuCfcUfguua 1273 GCCUAACAGGAGAUCAUAAGA 1632 auL96gsgsc AC AD-1136154.1 usasacagGfaGfAfUfcauaagaa 915asGfsuucUfuaugaucUfcCfuguuasgs 1274 CCUAACAGGAGAUCAUAAGAA 1633 cuL96 gCC AD-1136155.1 asascaggAfgAfUfCfauaagaac 916asGfsgudTc(Tgn)uaugauCfuCfcugu 1275 CUAACAGGAGAUCAUAAGAAC 1634 cuL96usasg CU AD-1136156.1 cscsuccgCfaCfAfGfauauuguc 917asUfsgacAfauaucugUfgCfggaggsus 1276 AACCUCCGCACAGAUAUUGUC 1635 auL96 uAU AD-1136157.1 csusccgcAfcAfGfAfuauuguca 918asAfsugaCfaauaucuGfuGfcggagsgs 1277 ACCUCCGCACAGAUAUUGUCA 1636 uuL96 uUG AD-1136158.1 ususggcuCfcAfGfUfcuaaaccc 919asAfsggdGu(Tgn)uagacuGfgAfgcca 1278 UGUUGGCUCCAGUCUAAACCC 1637 uuL96ascsa UG AD-1136159.1 ususccugGfcUfGfCfuucuaucu 920asAfsagaUfagaagcaGfcCfaggaasgs 1279 UCUUCCUGGCUGCUUCUAUCU 1638 uuL96 aUC AD-1136160.1 uscscuggCfuGfCfUfucuaucuu 921asGfsaagAfuagaagcAfgCfcaggasas 1280 CUUCCUGGCUGCUUCUAUCUU 1639 cuL96 gCU AD-1136161.1 cscsuggcUfgCfUfUfcuaucuuc 922asAfsgaaGfauagaagCfaGfccaggsas 1281 UUCCUGGCUGCUUCUAUCUUC 1640 uuL96 aUU AD-1136162.1 csusggcuGfcUfUfCfuaucuucu 923asAfsagaAfgauagaaGfcAfgccagsgs 1282 UCCUGGCUGCUUCUAUCUUCU 1641 uuL96 aUU AD-1136163.1 usgsgcugCfuUfCfUfaucuucuu 924asAfsaagAfagauagaAfgCfagccasgs 1283 CCUGGCUGCUUCUAUCUUCUU 1642 uuL96 gUG AD-1136164.1 gscsugcuUfcUfAfUfcuucuuug 925asGfscaaAfgaagauaGfaAfgcagcscs 1284 UGGCUGCUUCUAUCUUCUUUG 1643 cuL96 aCC AD-1136165.1 csusgcuuCfuAfUfCfuucuuugc 926asGfsgcaAfagaagauAfgAfagcagscs 1285 GGCUGCUUCUAUCUUCUUUGC 1644 cuL96 cCA AD-1136166.1 usgscuucUfaUfCfUfucuuugcc 927asUfsggdCa(Agn)agaagaUfaGfaagc 1286 GCUGCUUCUAUCUUCUUUGCC 1645 auL96asgsc AU AD-1136167.1 gscsuucuAfuCfUfUfcuuugcca 928asAfsugdGc(Agn)aagaagAfuAfgaag 1287 CUGCUUCUAUCUUCUUUGCCA 1646 uuL96csasg UC AD-1136168.1 csusucuaUfcUfUfCfuuugccau 929asGfsaudGg(C2p)aaagaaGfaUfagaa 1288 UGCUUCUAUCUUCUUUGCCAU 1647 cuL96gscsa CA AD-1136169.1 ususcuauCfuUfCfUfuugccauc 930asUfsgadTg(G2p)caaagaAfgAfuaga 1289 GCUUCUAUCUUCUUUGCCAUC 1648 auL96asgsc AA AD-1136170.1 uscsuaucUfuCfUfUfugccauca 931asUfsugdAu(G2p)gcaaagAfaGfauag 1290 CUUCUAUCUUCUUUGCCAUCA 1649 auL96asasg AA AD-1136171.1 csusaucuUfcUfUfUfgccaucaa 932asUfsuudGa(Tgn)ggcaaaGfaAfgaua 1291 UUCUAUCUUCUUUGCCAUCAA 1650 auL96gsasa AG AD-1136172.1 usasucuuCfuUfUfGfccaucaaa 933asCfsuuuGfauggcaaAfgAfagauasgs 1292 UCUAUCUUCUUUGCCAUCAAA 1651 guL96 aGA AD-1136173.1 asuscuucUfuUfGfCfcaucaaag 934asUfscudTu(G2p)auggcaAfaGfaaga 1293 CUAUCUUCUUUGCCAUCAAAG 1652 auL96usasg AU AD-1136174.1 csusucuuUfgCfCfAfucaaagau 935asCfsaucUfuugauggCfaAfagaagsas 1294 AUCUUCUUUGCCAUCAAAGAU 1653 guL96 uGC AD-1136175.1 gsasgcucAfgCfAfCfacagguaa 936asAfsuudAc(C2p)ugugugCfuGfagcu 1295 UCGAGCUCAGCACACAGGUAA 1654 uuL96csgsa UA AD-1136176.1 csuscagcAfcAfCfAfgguaauaa 937asGfsuuaUfuaccuguGfuGfcugagscs 1296 AGCUCAGCACACAGGUAAUAA 1655 cuL96 uCG AD-1136177.1 uscsagcaCfaCfAfGfguaauaac 938asCfsguuAfuuaccugUfgUfgcugasgs 1297 GCUCAGCACACAGGUAAUAAC 1656 guL96 cGU AD-1136178.1 csasgcacAfcAfGfGfuaauaacg 939asAfscguUfauuaccuGfuGfugcugsas 1298 CUCAGCACACAGGUAAUAACG 1657 uuL96 gUG AD-1136179.1 ascsagguAfaUfAfAfcgugaagg 940asUfsccdTu(C2p)acguuaUfuAfccug 1299 ACACAGGUAAUAACGUGAAGG 1658 auL96usgsu AA AD-1136180.1 csasgguaAfuAfAfCfgugaagga 941asUfsucdCu(Tgn)cacguuAfuUfaccu 1300 CACAGGUAAUAACGUGAAGGA 1659 auL96gsusg AC AD-1136181.1 asgsguaaUfaAfCfGfugaaggaa 942asGfsuudCc(Tgn)ucacguUfaUfuacc 1301 ACAGGUAAUAACGUGAAGGAA 1660 cuL96usgsu CU AD-1136182.1 asusaacgUfgAfAfGfgaacucuu 943asGfsaadGa(G2p)uuccuuCfaCfguua 1302 UAAUAACGUGAAGGAACUCUU 1661 cuL96ususa CC AD-1136183.1 usasacguGfaAfGfGfaacucuuc 944asGfsgadAg(Agn)guuccuUfcAfcguu 1303 AAUAACGUGAAGGAACUCUUC 1662 cuL96asusu CG AD-1136184.1 cscscagaAfaAfCfUfgcaaaccc 945asAfsggdGu(Tgn)ugcaguUfuUfcugg 1304 GUCCCAGAAAACUGCAAACCC 1663 uuL96gsasc UG AD-1136185.1 csascagaAfcAfUfGfgaucuauu 946asUfsaadTa(G2p)auccauGfuUfcugu 1305 ACCACAGAACAUGGAUCUAUU 1664 auL96gsgsu AA AD-1136186.1 ascsagaaCfaUfGfGfaucuauua 947asUfsuaaUfagauccaUfgUfucugusgs 1306 CCACAGAACAUGGAUCUAUUA 1665 auL96 gAA AD-1136187.1 csasgaacAfuGfGfAfucuauuaa 948asUfsuuaAfuagauccAfuGfuucugsus 1307 CACAGAACAUGGAUCUAUUAA 1666 auL96 gAG AD-1136188.1 asgsaacaUfgGfAfUfcuauuaaa 949asCfsuuuAfauagaucCfaUfguucusgs 1308 ACAGAACAUGGAUCUAUUAAA 1667 guL96 uGU AD-1136189.1 gsasacauGfgAfUfCfuauuaaag 950asAfscuuUfaauagauCfcAfuguucsus 1309 CAGAACAUGGAUCUAUUAAAG 1668 uuL96 gUC AD-1136190.1 asascaugGfaUfCfUfauuaaagu 951asGfsacuUfuaauagaUfcCfauguuscs 1310 AGAACAUGGAUCUAUUAAAGU 1669 cuL96 uCA AD-1136191.1 ascsauggAfuCfUfAfuuaaaguc 952asUfsgadCu(Tgn)uaauagAfuCfcaug 1311 GAACAUGGAUCUAUUAAAGUC 1670 auL96ususc AC AD-1136192.1 csasuggaUfcUfAfUfuaaaguca 953asGfsugdAc(Tgn)uuaauaGfaUfccau 1312 AACAUGGAUCUAUUAAAGUCA 1671 cuL96gsusu CA AD-1136193.1 asusggauCfuAfUfUfaaagucac 954asUfsgudGa(C2p)uuuaauAfgAfucca 1313 ACAUGGAUCUAUUAAAGUCAC 1672 auL96usgsu AG AD-1136194.1 usgsgaucUfaUfUfAfaagucaca 955asCfsugdTg(Agn)cuuuaaUfaGfaucc 1314 CAUGGAUCUAUUAAAGUCACA 1673 guL96asusg GA AD-1136195.1 gsgsaucuAfuUfAfAfagucacag 956asUfscudGu(G2p)acuuuaAfuAfgauc 1315 AUGGAUCUAUUAAAGUCACAG 1674 auL96csasu AA AD-1136196.1 gsasucuaUfuAfAfAfgucacaga 957asUfsucdTg(Tgn)gacuuuAfaUfagau 1316 UGGAUCUAUUAAAGUCACAGA 1675 auL96cscsa AU AD-1136197.1 ascsaaugAfuAfAfGfcaaauuca 958asUfsugaAfuuugcuuAfuCfauugusgs 1317 ACACAAUGAUAAGCAAAUUCA 1676 auL96 uAA AD-1136198.1 csasaugaUfaAfGfCfaaauucaa 959asUfsuudGa(Agn)uuugcuUfaUfcauu 1318 CACAAUGAUAAGCAAAUUCAA 1677 auL96gsusg AA AD-1136199.1 asusgauaAfgCfAfAfauucaaaa 960asGfsuudTu(G2p)aauuugCfuUfauca 1319 CAAUGAUAAGCAAAUUCAAAA 1678 cuL96ususg CU AD-1136200.1 asusgccuAfaAfUfGfgugaauau 961asCfsauaUfucaccauUfuAfggcausas 1320 UUAUGCCUAAAUGGUGAAUAU 1679 guL96 aGC AD-1136201.1 usgsccuaAfaUfGfGfugaauaug 962asGfscauAfuucaccaUfuUfaggcasus 1321 UAUGCCUAAAUGGUGAAUAUG 1680 cuL96 aCA AD-1136202.1 gscscuaaAfuGfGfUfgaauaugc 963asUfsgcdAu(Agn)uucaccAfuUfuagg 1322 AUGCCUAAAUGGUGAAUAUGC 1681 auL96csasu AA AD-1136203.1 cscsuaaaUfgGfUfGfaauaugca 964asUfsugdCa(Tgn)auucacCfaUfuuag 1323 UGCCUAAAUGGUGAAUAUGCA 1682 auL96gscsa AU AD-1136204.1 csusaaauGfgUfGfAfauaugcaa 965asAfsuudGc(Agn)uauucaCfcAfuuua 1324 GCCUAAAUGGUGAAUAUGCAA 1683 uuL96gsgsc UU AD-1136205.1 asasugguGfaAfUfAfugcaauua 966asCfsuaaUfugcauauUfcAfccauusus 1325 UAAAUGGUGAAUAUGCAAUUA 1684 guL96 aGG AD-1136206.1 csgsggaaGfgGfUfUfugugcuau 967asAfsaudAg(C2p)acaaacCfcUfuccc 1326 GUCGGGAAGGGUUUGUGCUAU 1685 uuL96gsasc UC AD-1136207.1 gsgsgaagGfgUfUfUfgugcuauu 968asGfsaadTa(G2p)cacaaaCfcCfuucc 1327 UCGGGAAGGGUUUGUGCUAUU 1686 cuL96csgsa CC AD-1136208.1 gsgsaaggGfuUfUfGfugcuauuc 969asGfsgaaUfagcacaaAfcCfcuuccscs 1328 CGGGAAGGGUUUGUGCUAUUC 1687 cuL96 gCC AD-1136209.1 gsusauaaCfcUfCfAfaguucuga 970asAfsucaGfaacuugaGfgUfuauacsas 1329 CUGUAUAACCUCAAGUUCUGA 1688 uuL96 gUG AD-1136210.1 usasuaacCfuCfAfAfguucugau 971asCfsaudCa(G2p)aacuugAfgGfuuau 1330 UGUAUAACCUCAAGUUCUGAU 1689 guL96ascsa GG AD-1136211.1 cscsucaaGfuUfCfUfgauggugu 972asGfsacdAc(C2p)aucagaAfcUfugag 1331 AACCUCAAGUUCUGAUGGUGU 1690 cuL96gsusu CU AD-1136212.1 csasaguuCfuGfAfUfggugucug 973asAfscadGa(C2p)accaucAfgAfacuu 1332 CUCAAGUUCUGAUGGUGUCUG 1691 uuL96gsasg UC AD-1136213.1 cscsacaaAfcCfUfCfuagaagcu 974asAfsagdCu(Tgn)cuagagGfuUfugug 1333 UCCCACAAACCUCUAGAAGCU 1692 uuL96gsgsa UA AD-1136214.1 ascsaaacCfuCfUfAfgaagcuua 975asUfsuadAg(C2p)uucuagAfgGfuuug 1334 CCACAAACCUCUAGAAGCUUA 1693 auL96usgsg AA AD-1136215.1 asasaccuCfuAfGfAfagcuuaaa 976asGfsuuuAfagcuucuAfgAfgguuusgs 1335 ACAAACCUCUAGAAGCUUAAA 1694 cuL96 uCC AD-1136216.1 asasccucUfaGfAfAfgcuuaaac 977asGfsguuUfaagcuucUfaGfagguusus 1336 CAAACCUCUAGAAGCUUAAAC 1695 cuL96 gCG AD-1136217.1 usgsgccuUfcAfAfAfccaaugaa 978asGfsuucAfuugguuuGfaAfggccasgs 1337 CCUGGCCUUCAAACCAAUGAA 1696 cuL96 gCA AD-1136218.1 gsgsccuuCfaAfAfCfcaaugaac 979asUfsgudTc(Agn)uugguuUfgAfaggc 1338 CUGGCCUUCAAACCAAUGAAC 1697 auL96csasg AG AD-1136219.1 gscscuucAfaAfCfCfaaugaaca 980asCfsuguUfcauugguUfuGfaaggcscs 1339 UGGCCUUCAAACCAAUGAACA 1698 guL96 aGC AD-1136220.1 uscsaaacCfaAfUfGfaacagcaa 981asUfsuudGc(Tgn)guucauUfgGfuuug 1340 CUUCAAACCAAUGAACAGCAA 1699 auL96asasg AG AD-1136221.1 gsasacagCfaAfAfGfcauaaccu 982asAfsaggUfuaugcuuUfgCfuguucsas 1341 AUGAACAGCAAAGCAUAACCU 1700 uuL96 uUG AD-1136222.1 asascagcAfaAfGfCfauaaccuu 983asCfsaadGg(Tgn)uaugcuUfuGfcugu 1342 UGAACAGCAAAGCAUAACCUU 1701 guL96uscsa GA AD-1136223.1 asgscaaaGfcAfUfAfaccuugaa 984asAfsuucAfagguuauGfcUfuugcusgs 1343 ACAGCAAAGCAUAACCUUGAA 1702 uuL96 uUC AD-1136224.1 gscsaaagCfaUfAfAfccuugaau 985asGfsaudTc(Agn)agguuaUfgCfuuug 1344 CAGCAAAGCAUAACCUUGAAU 1703 cuL96csusg CU AD-1136225.1 csasaagcAfuAfAfCfcuugaauc 986asAfsgadTu(C2p)aagguuAfuGfcuuu 1345 AGCAAAGCAUAACCUUGAAUC 1704 uuL96gscsu UA AD-1136226.1 gscsauaaCfcUfUfGfaaucuaua 987asGfsuauAfgauucaaGfgUfuaugcsus 1346 AAGCAUAACCUUGAAUCUAUA 1705 cuL96 uCU AD-1136227.1 csasuaacCfuUfGfAfaucuauac 988asAfsguaUfagauucaAfgGfuuaugscs 1347 AGCAUAACCUUGAAUCUAUAC 1706 uuL96 uUC AD-1136228.1 asusaaccUfuGfAfAfucuauacu 989asGfsaguAfuagauucAfaGfguuausgs 1348 GCAUAACCUUGAAUCUAUACU 1707 cuL96 cCA AD-1136229.1 usasaccuUfgAfAfUfcuauacuc 990asUfsgadGu(Agn)uagauuCfaAfgguu 1349 CAUAACCUUGAAUCUAUACUC 1708 auL96asusg AA AD-1136230.1 asasccuuGfaAfUfCfuauacuca 991asUfsugdAg(Tgn)auagauUfcAfaggu 1350 AUAACCUUGAAUCUAUACUCA 1709 auL96usasu AA AD-1136231.1 ascscuugAfaUfCfUfauacucaa 992asUfsuudGa(G2p)uauagaUfuCfaagg 1351 UAACCUUGAAUCUAUACUCAA 1710 auL96ususa AU AD-1136232.1 cscsuugaAfuCfUfAfuacucaaa 993asAfsuudTg(Agn)guauagAfuUfcaag 1352 AACCUUGAAUCUAUACUCAAA 1711 uuL96gsusu UU AD-1136233.1 asasucuaUfaCfUfCfaaauuuug 994asGfscaaAfauuugagUfaUfagauuscs 1353 UGAAUCUAUACUCAAAUUUUG 1712 cuL96 aCA AD-1136234.1 asuscuauAfcUfCfAfaauuuugc 995asUfsgcaAfaauuugaGfuAfuagausus 1354 GAAUCUAUACUCAAAUUUUGC 1713 auL96 cAA AD-1136235.1 gsgsuuaaAfuCfCfUfcuaaccau 996asGfsaudGg(Tgn)uagaggAfuUfuaac 1355 AAGGUUAAAUCCUCUAACCAU 1714 cuL96csusu CU AD-1136236.1 usasaaucCfuCfUfAfaccaucuu 997asAfsaadGa(Tgn)gguuagAfgGfauuu 1356 GUUAAAUCCUCUAACCAUCUU 1715 uuL96asasc UG AD-1136237.1 asasauccUfcUfAfAfccaucuuu 998asCfsaaaGfaugguuaGfaGfgauuusas 1357 UUAAAUCCUCUAACCAUCUUU 1716 guL96 aGA AD-1136238.1 asasuccuCfuAfAfCfcaucuuug 999asUfscaaAfgaugguuAfgAfggauusus 1358 UAAAUCCUCUAACCAUCUUUG 1717 auL96 aAA AD-1136239.1 asusccucUfaAfCfCfaucuuuga 1000asUfsucaAfagaugguUfaGfaggausus 1359 AAAUCCUCUAACCAUCUUUGA 1718 auL96 uAU AD-1136240.1 uscscucuAfaCfCfAfucuuugaa 1001asAfsuucAfaagauggUfuAfgaggasus 1360 AAUCCUCUAACCAUCUUUGAA 1719 uuL96 uUC AD-1136241.1 cscsucuaAfcCfAfUfcuuugaau 1002asGfsaudTc(Agn)aagaugGfuUfagag 1361 AUCCUCUAACCAUCUUUGAAU 1720 cuL96gsasu CA AD-1136242.1 csuscuaaCfcAfUfCfuuugaauc 1003asUfsgadTu(C2p)aaagauGfgUfuaga 1362 UCCUCUAACCAUCUUUGAAUC 1721 auL96gsgsa AU AD-1136243.1 uscsuaacCfaUfCfUfuugaauca 1004asAfsugaUfucaaagaUfgGfuuagasgs 1363 CCUCUAACCAUCUUUGAAUCA 1722 uuL96 gUU AD-1136244.1 csusaaccAfuCfUfUfugaaucau 1005asAfsaudGa(Tgn)ucaaagAfuGfguua 1364 CUCUAACCAUCUUUGAAUCAU 1723 uuL96gsasg UG AD-1136245.1 usasaccaUfcUfUfUfgaaucauu 1006asCfsaadTg(Agn)uucaaaGfaUfgguu 1365 UCUAACCAUCUUUGAAUCAUU 1724 guL96asgsa GG AD-1136246.1 asasccauCfuUfUfGfaaucauug 1007asCfscaaUfgauucaaAfgAfugguusas 1366 CUAACCAUCUUUGAAUCAUUG 1725 guL96 gGA AD-1136247.1 cscsaucuUfuGfAfAfucauugga 1008asUfsuccAfaugauucAfaAfgauggsus 1367 AACCAUCUUUGAAUCAUUGGA 1726 auL96 uAA AD-1136248.1 csasucuuUfgAfAfUfcauuggaa 1009asUfsuudCc(Agn)augauuCfaAfagau 1368 ACCAUCUUUGAAUCAUUGGAA 1727 auL96gsgsu AG AD-1136249.1 asuscuuuGfaAfUfCfauuggaaa 1010asCfsuudTc(C2p)aaugauUfcAfaaga 1369 CCAUCUUUGAAUCAUUGGAAA 1728 guL96usgsg GA AD-1136250.1 csusuugaAfuCfAfUfuggaaaga 1011asUfsucdTu(Tgn)ccaaugAfuUfcaaa 1370 AUCUUUGAAUCAUUGGAAAGA 1729 auL96gsasu AU AD-1136251.1 gsasaagaAfuAfAfAfgaaugaaa 1012asGfsuudTc(Agn)uucuuuAfuUfcuuu 1371 UGGAAAGAAUAAAGAAUGAAA 1730 cuL96cscsa CA AD-1136252.1 asasagaaUfaAfAfGfaaugaaac 1013asUfsgudTu(C2p)auucuuUfaUfucuu 1372 GGAAAGAAUAAAGAAUGAAAC 1731 auL96uscsc AA AD-1136253.1 gsasaugaAfaCfAfAfauucaagg 1014asAfsccuUfgaauuugUfuUfcauucsus 1373 AAGAAUGAAACAAAUUCAAGG 1732 uuL96 uUU AD-1136254.1 asusgaaaCfaAfAfUfucaagguu 1015asUfsaadCc(Tgn)ugaauuUfgUfuuca 1374 GAAUGAAACAAAUUCAAGGUU 1733 auL96ususc AA AD-1136255.1 asascaaaUfuCfAfAfgguuaauu 1016asCfsaauUfaaccuugAfaUfuuguusus 1375 GAAACAAAUUCAAGGUUAAUU 1734 guL96 cGG AD-1136256.1 usgsaagcUfgCfAfUfaaagcaag 1017asUfscudTg(C2p)uuuaugCfaGfcuuc 1376 UGUGAAGCUGCAUAAAGCAAG 1735 auL96ascsa AU AD-1136257.1 asasgcugCfaUfAfAfagcaagau 1018asAfsaudCu(Tgn)gcuuuaUfgCfagcu 1377 UGAAGCUGCAUAAAGCAAGAU 1736 uuL96uscsa UA AD-1136258.1 asgscugcAfuAfAfAfgcaagauu 1019asUfsaadTc(Tgn)ugcuuuAfuGfcagc 1378 GAAGCUGCAUAAAGCAAGAUU 1737 auL96USUSC AC AD-1136259.1 gscsugcaUfaAfAfGfcaagauua 1020asGfsuadAu(C2p)uugcuuUfaUfgcag 1379 AAGCUGCAUAAAGCAAGAUUA 1738 cuL96csusu CU AD-1136260.1 csusgcauAfaAfGfCfaagauuac 1021asAfsguaAfucuugcuUfuAfugcagscs 1380 AGCUGCAUAAAGCAAGAUUAC 1739 uuL96 uUC AD-1136261.1 usgscauaAfaGfCfAfagauuacu 1022asGfsaguAfaucuugcUfuUfaugcasgs 1381 GCUGCAUAAAGCAAGAUUACU 1740 cuL96 cCU AD-1136262.1 csasuaaaGfcAfAfGfauuacucu 1023asUfsagdAg(Tgn)aaucuuGfcUfuuau 1382 UGCAUAAAGCAAGAUUACUCU 1741 auL96gscsa AU AD-1136263.1 usasaagcAfaGfAfUfuacucuau 1024asUfsaudAg(Agn)guaaucUfuGfcuuu 1383 CAUAAAGCAAGAUUACUCUAU 1742 auL96asusg AA AD-1136264.1 asusacaaAfaAfUfCfcaaccaac 1025asAfsgudTg(G2p)uuggauUfuUfugua 1384 UAAUACAAAAAUCCAACCAAC 1743 uuL96ususa UC AD-1136265.1 usascaaaAfaUfCfCfaaccaacu 1026asGfsagdTu(G2p)guuggaUfuUfuugu 1385 AAUACAAAAAUCCAACCAACU 1744 cuL96asusu CA AD-1136266.1 ascsaaaaAfuCfCfAfaccaacuc 1027asUfsgadGu(Tgn)gguuggAfuUfuuug 1386 AUACAAAAAUCCAACCAACUC 1745 auL96usasu AA AD-1136267.1 csasaaaaUfcCfAfAfccaacuca 1028asUfsugdAg(Tgn)ugguugGfaUfuuuu 1387 UACAAAAAUCCAACCAACUCA 1746 auL96gsusa AU AD-1136268.1 asasaaauCfcAfAfCfcaacucaa 1029asAfsuudGa(G2p)uugguuGfgAfuuuu 1388 ACAAAAAUCCAACCAACUCAA 1747 uuL96usgsu UU AD-1136269.1 asasaaucCfaAfCfCfaacucaau 1030asAfsaudTg(Agn)guugguUfgGfauuu 1389 CAAAAAUCCAACCAACUCAAU 1748 uuL96ususg UA AD-1136270.1 asasauccAfaCfCfAfacucaauu 1031asUfsaadTu(G2p)aguuggUfuGfgauu 1390 AAAAAUCCAACCAACUCAAUU 1749 auL96ususu AU AD-1136271.1 asasuccaAfcCfAfAfcucaauua 1032asAfsuaaUfugaguugGfuUfggauusus 1391 AAAAUCCAACCAACUCAAUUA 1750 uuL96 uUU AD-1136272.1 asusccaaCfcAfAfCfucaauuau 1033asAfsauaAfuugaguuGfgUfuggausus 1392 AAAUCCAACCAACUCAAUUAU 1751 uuL96 uUG AD-1136273.1 uscscaacCfaAfCfUfcaauuauu 1034asCfsaauAfauugaguUfgGfuuggasus 1393 AAUCCAACCAACUCAAUUAUU 1752 guL96 uGA AD-1136274.1 cscsaaccAfaCfUfCfaauuauug 1035asUfscaaUfaauugagUfuGfguuggsas 1394 AUCCAACCAACUCAAUUAUUG 1753 auL96 uAG AD-1136275.1 csasaccaAfcUfCfAfauuauuga 1036asCfsucaAfuaauugaGfuUfgguugsgs 1395 UCCAACCAACUCAAUUAUUGA 1754 guL96 aGC AD-1136276.1 asasccaaCfuCfAfAfuuauugag 1037asGfscucAfauaauugAfgUfugguusgs 1396 CCAACCAACUCAAUUAUUGAG 1755 cuL96 gCA AD-1136277.1 ascscaacUfcAfAfUfuauugagc 1038asUfsgcdTc(Agn)auaauuGfaGfuugg 1397 CAACCAACUCAAUUAUUGAGC 1756 auL96ususg AC AD-1136278.1 cscsaacuCfaAfUfUfauugagca 1039asGfsugdCu(C2p)aauaauUfgAfguug 1398 AACCAACUCAAUUAUUGAGCA 1757 cuL96gsusu CG AD-1136279.1 csgsuacaAfuGfUfUfcuagauuu 1040asGfsaadAu(C2p)uagaacAfuUfguac 1399 CACGUACAAUGUUCUAGAUUU 1758 cuL96gsusg CU AD-1136280.1 gsusacaaUfgUfUfCfuagauuuc 1041asAfsgaaAfucuagaaCfaUfuguacsgs 1400 ACGUACAAUGUUCUAGAUUUC 1759 uuL96 uUU AD-1136281.1 usascaauGfuUfCfUfagauuucu 1042asAfsagaAfaucuagaAfcAfuuguascs 1401 CGUACAAUGUUCUAGAUUUCU 1760 uuL96 gUU AD-1136282.1 ascsaaugUfuCfUfAfgauuucuu 1043asAfsaagAfaaucuagAfaCfauugusas 1402 GUACAAUGUUCUAGAUUUCUU 1761 uuL96 cUC AD-1136283.1 csasauguUfcUfAfGfauuucuuu 1044asGfsaaaGfaaaucuaGfaAfcauugsus 1403 UACAAUGUUCUAGAUUUCUUU 1762 cuL96 aCC AD-1136284.1 asasuguuCfuAfGfAfuuucuuuc 1045asGfsgaaAfgaaaucuAfgAfacauusgs 1404 ACAAUGUUCUAGAUUUCUUUC 1763 cuL96 uCC AD-1136285.1 gsusucuaGfaUfUfUfcuuucccu 1046asAfsagdGg(Agn)aagaaaUfcUfagaa 1405 AUGUUCUAGAUUUCUUUCCCU 1764 uuL96csasu UC AD-1136286.1 ususcuagAfuUfUfCfuuucccuu 1047asGfsaadGg(G2p)aaagaaAfuCfuaga 1406 UGUUCUAGAUUUCUUUCCCUU 1765 cuL96ascsa CC AD-1136287.1 uscsuuuaCfaUfUfUfgaagccaa 1048asUfsuudGg(C2p)uucaaaUfgUfaaag 1407 AAUCUUUACAUUUGAAGCCAA 1766 auL96asusu AG AD-1136288.1 csusuuacAfuUfUfGfaagccaaa 1049asCfsuudTg(G2p)cuucaaAfuGfuaaa 1408 AUCUUUACAUUUGAAGCCAAA 1767 guL96gsasu GU AD-1136289.1 ususuacaUfuUfGfAfagccaaag 1050asAfscudTu(G2p)gcuucaAfaUfguaa 1409 UCUUUACAUUUGAAGCCAAAG 1768 uuL96asgsa UA AD-1136290.1 ascsauuuGfaAfGfCfcaaaguaa 1051asAfsuudAc(Tgn)uuggcuUfcAfaaug 1410 UUACAUUUGAAGCCAAAGUAA 1769 uuL96usasa UU AD-1136291.1 asusuugaAfgCfCfAfaaguaauu 1052asAfsaauUfacuuuggCfuUfcaaausgs 1411 ACAUUUGAAGCCAAAGUAAUU 1770 uuL96 uUC AD-1136292.1 gsasagccAfaAfGfUfaauuucca 1053asGfsuggAfaauuacuUfuGfgcuucsas 1412 UUGAAGCCAAAGUAAUUUCCA 1771 cuL96 aCC AD-1136293.1 asasgccaAfaGfUfAfauuuccac 1054asGfsgudGg(Agn)aauuacUfuUfggcu 1413 UGAAGCCAAAGUAAUUUCCAC 1772 cuL96uscsa CU AD-1136294.1 cscsaaagUfaAfUfUfuccaccua 1055asCfsuadGg(Tgn)ggaaauUfaCfuuug 1414 AGCCAAAGUAAUUUCCACCUA 1773 guL96gscsu GA AD-1136295.1 csasaaguAfaUfUfUfccaccuag 1056asUfscudAg(G2p)uggaaaUfuAfcuuu 1415 GCCAAAGUAAUUUCCACCUAG 1774 auL96gsgsc AA AD-1136296.1 asasaguaAfuUfUfCfcaccuaga 1057asUfsucdTa(G2p)guggaaAfuUfacuu 1416 CCAAAGUAAUUUCCACCUAGA 1775 auL96usgsg AA AD-1136297.1 gscsaagaUfcUfUfGfucucugaa 1058asCfsuucAfgagacaaGfaUfcuugcsus 1417 GAGCAAGAUCUUGUCUCUGAA 1776 guL96 cGA AD-1136298.1 asgsguagAfaGfAfGfccaagaag 1059asGfscudTc(Tgn)uggcucUfuCfuacc 1418 AGAGGUAGAAGAGCCAAGAAG 1777 cuL96uscsu CC AD-1136299.1 csusagcuCfuGfUfCfucuucugu 1060asGfsacaGfaagagacAfgAfgcuagsgs 1419 UCCUAGCUCUGUCUCUUCUGU 1778 cuL96 aCU AD-1136300.1 usasgcucUfgUfCfUfcuucuguc 1061asAfsgadCa(G2p)aagagaCfaGfagcu 1420 CCUAGCUCUGUCUCUUCUGUC 1779 uuL96asgsg uc AD-1136301.1 asgscucuGfuCfUfCfuucugucu 1062asGfsagdAc(Agn)gaagagAfcAfgagc 1421 CUAGCUCUGUCUCUUCUGUCU 1780 cuL96usasg CU AD-1136302.1 asuscuuuGfuGfAfUfcuuggacu 1063asCfsaguCfcaagaucAfcAfaagausas 1422 CUAUCUUUGUGAUCUUGGACU 1781 guL96 gGU AD-1136303.1 csusuccuGfuGfAfUfccauuuua 1064asGfsuaaAfauggaucAfcAfggaagsgs 1423 CCCUUCCUGUGAUCCAUUUUA 1782 cuL96 gCU AD-1136304.1 ususccugUfgAfUfCfcauuuuac 1065asAfsguaAfaauggauCfaCfaggaasgs 1424 CCUUCCUGUGAUCCAUUUUAC 1783 uuL96 gUG AD-1136305.1 uscscuguGfaUfCfCfauuuuacu 1066asCfsaguAfaaauggaUfcAfcaggasas 1425 CUUCCUGUGAUCCAUUUUACU 1784 guL96 gGC AD-1136306.1 cscsugugAfuCfCfAfuuuuacug 1067asGfscagUfaaaauggAfuCfacaggsas 1426 UUCCUGUGAUCCAUUUUACUG 1785 cuL96 aCA AD-1136307.1 csusgugaUfcCfAfUfuuuacugc 1068asUfsgcdAg(Tgn)aaaaugGfaUfcaca 1427 UCCUGUGAUCCAUUUUACUGC 1786 auL96gsgsa AA AD-1136308.1 csusauaaAfuAfUfAfugaccuga 1069asUfsucdAg(G2p)ucauauAfuUfuaua 1428 GCCUAUAAAUAUAUGACCUGA 1787 auL96gsgsc AA AD-1136309.1 usgsaccuGfaAfAfAfcuccaguu 1070asUfsaadCu(G2p)gaguuuUfcAfgguc 1429 UAUGACCUGAAAACUCCAGUU 1788 auL96asusa AC AD-1136310.1 gsasccugAfaAfAfCfuccaguua 1071asGfsuadAc(Tgn)ggaguuUfuCfaggu 1430 AUGACCUGAAAACUCCAGUUA 1789 cuL96csasu CA AD-1136311.1 asasggauCfuGfCfAfgcuaucua 1072asUfsuadGa(Tgn)agcugcAfgAfuccu 1431 UAAAGGAUCUGCAGCUAUCUA 1790 auL96ususa AG AD-1136312.1 asgsgaucUfgCfAfGfcuaucuaa 1073asCfsuuaGfauagcugCfaGfauccusus 1432 AAAGGAUCUGCAGCUAUCUAA 1791 guL96 uGG AD-1136313.1 gscsuaucUfaAfGfGfcuugguuu 1074asAfsaaaCfcaagccuUfaGfauagcsus 1433 CAGCUAUCUAAGGCUUGGUUU 1792 uuL96 gUC AD-1136314.1 csusaucuAfaGfGfCfuugguuuu 1075asGfsaaaAfccaagccUfuAfgauagscs 1434 AGCUAUCUAAGGCUUGGUUUU 1793 cuL96 uCU AD-1136315.1 asuscuaaGfgCfUfUfgguuuucu 1076asAfsagaAfaaccaagCfcUfuagausas 1435 CUAUCUAAGGCUUGGUUUUCU 1794 uuL96 gUA AD-1136316.1 uscsuaagGfcUfUfGfguuuucuu 1077asUfsaadGa(Agn)aaccaaGfcCfuuag 1436 UAUCUAAGGCUUGGUUUUCUU 1795 auL96asusa AC AD-1136317.1 csusaaggCfuUfGfGfuuuucuua 1078asGfsuaaGfaaaaccaAfgCfcuuagsas 1437 AUCUAAGGCUUGGUUUUCUUA 1796 cuL96 uCU AD-1136318.1 usasaggcUfuGfGfUfuuucuuac 1079asAfsguaAfgaaaaccAfaGfccuuasgs 1438 UCUAAGGCUUGGUUUUCUUAC 1797 uuL96 aUG AD-1136319.1 gsgscuugGfuUfUfUfcuuacugu 1080asGfsacdAg(Tgn)aagaaaAfcCfaagc 1439 AAGGCUUGGUUUUCUUACUGU 1798 cuL96csusu CA AD-1136320.1 gscsuuggUfuUfUfCfuuacuguc 1081asUfsgadCa(G2p)uaagaaAfaCfcaag 1440 AGGCUUGGUUUUCUUACUGUC 1799 auL96cscsu AU AD-1136321.1 csusugguUfuUfCfUfuacuguca 1082asAfsugdAc(Agn)guaagaAfaAfccaa 1441 GGCUUGGUUUUCUUACUGUCA 1800 uuL96gscsc UA AD-1136322.1 ususgguuUfuCfUfUfacugucau 1083asUfsaudGa(C2p)aguaagAfaAfacca 1442 GCUUGGUUUUCUUACUGUCAU 1801 auL96asgsc AU AD-1136323.1 usgsguuuUfcUfUfAfcugucaua 1084asAfsuadTg(Agn)caguaaGfaAfaacc 1443 CUUGGUUUUCUUACUGUCAUA 1802 uuL96asasg UG AD-1136324.1 gsgsuuuuCfuUfAfCfugucauau 1085asCfsauaUfgacaguaAfgAfaaaccsas 1444 UUGGUUUUCUUACUGUCAUAU 1803 guL96 aGA AD-1136325.1 ususuucuUfaCfUfGfucauauga 1086asAfsucaUfaugacagUfaAfgaaaascs 1445 GGUUUUCUUACUGUCAUAUGA 1804 uuL96 cUA AD-1136326.1 ususucuuAfcUfGfUfcauaugau 1087asUfsaucAfuaugacaGfuAfagaaasas 1446 GUUUUCUUACUGUCAUAUGAU 1805 auL96 cAC AD-1136327.1 uscsuuacUfgUfCfAfuaugauac 1088asGfsgudAu(C2p)auaugaCfaGfuaag 1447 UUUCUUACUGUCAUAUGAUAC 1806 cuL96asasa CU AD-1136328.1 csusuacuGfuCfAfUfaugauacc 1089asAfsgguAfucauaugAfcAfguaagsas 1448 UUCUUACUGUCAUAUGAUACC 1807 uuL96 aUG AD-1136329.1 uscsugcgGfuUfGfGfuaaagaga 1090asUfsucdTc(Tgn)uuaccaAfcCfgcag 1449 UUUCUGCGGUUGGUAAAGAGA 1808 auL96asasa AC AD-1136330.1 usgsgugaUfuUfAfAfucccuaca 1091asAfsugdTa(G2p)ggauuaAfaUfcacc 1450 AAUGGUGAUUUAAUCCCUACA 1809 uuL96asusu UG AD-1136331.1 gsgsugauUfuAfAfUfcccuacau 1092asCfsaugUfagggauuAfaAfucaccsas 1451 AUGGUGAUUUAAUCCCUACAU 1810 guL96 uGG AD-1136332.1 usgsuauaAfaUfCfCfaaccuucu 1093asCfsagaAfgguuggaUfuUfauacasgs 1452 ACUGUAUAAAUCCAACCUUCU 1811 guL96 uGC AD-1136333.1 gsusgcuuGfaUfGfUfcacuaaua 1094asUfsuadTu(Agn)gugacaUfcAfagca 1453 UGGUGCUUGAUGUCACUAAUA 1812 auL96cscsa AA AD-1136334.1 usgscuugAfuGfUfCfacuaauaa 1095asUfsuuaUfuagugacAfuCfaagcascs 1454 GGUGCUUGAUGUCACUAAUAA 1813 auL96 cAU AD-1136335.1 gscsuugaUfgUfCfAfcuaauaaa 1096asAfsuuuAfuuagugaCfaUfcaagcsas 1455 GUGCUUGAUGUCACUAAUAAA 1814 uuL96 cUG AD-1136336.1 asusgucaCfuAfAfUfaaaugaaa 1097asGfsuudTc(Agn)uuuauuAfgUfgaca 1456 UGAUGUCACUAAUAAAUGAAA 1815 cuL96uscsa CU AD-1136337.1 usgsucacUfaAfUfAfaaugaaac 1098asAfsgudTu(C2p)auuuauUfaGfugac 1457 GAUGUCACUAAUAAAUGAAAC 1816 uuL96asusc UG AD-1136338.1 usasauaaAfuGfAfAfacugucag 1099asGfscudGa(C2p)aguuucAfuUfuauu 1458 ACUAAUAAAUGAAACUGUCAG 1817 cuL96asgsu CU

TABLE 4 Xanthine Dehydrogenase dsRNA Agent In Vitro Single Dose Screensin Primary Human Hepatocytes PHH 500 nM 100 nM 10 nM % of Avg % of Avg %of Avg Message Message Message Duplex ID Remaining STDEV Remaining STDEVRemaining STDEV AD-1135979.1 78.6 8.5 81.4 6.5 68.6 25.5 AD-1135980.198.2 10.7 92.0 18.8 64.8 1.2 AD-1135981.1 79.5 14.5 92.4 19.2 60.7 9.1AD-1135982.1 66.7 6.7 80.3 6.8 67.5 4.1 AD-1135983.1 68.7 9.7 75.0 3.562.8 6.8 AD-1135984.1 69.4 14.9 66.0 5.0 68.3 13.1 AD-1135985.1 59.810.9 69.5 26.8 69.2 9.4 AD-1135986.1 94.2 30.7 54.0 21.9 58.0 7.8AD-1135987.1 29.0 5.3 47.3 10.4 44.4 7.8 AD-1135988.1 82.4 15.5 85.9 4.863.9 1.9 AD-1135989.1 36.6 8.6 58.0 10.7 52.9 3.2 AD-1135990.1 86.8 31.494.2 5.1 73.2 16.0 AD-1135991.1 36.0 6.7 45.8 9.5 54.3 9.2 AD-1135992.166.2 18.6 81.8 9.3 78.3 14.7 AD-1135993.1 68.7 16.2 56.9 17.0 62.7 7.4AD-1135994.1 60.5 21.8 71.3 22.4 70.5 18.1 AD-1135995.1 51.2 5.0 76.56.1 54.8 6.4 AD-1135996.1 38.6 8.3 56.5 8.9 54.8 7.4 AD-1135997.1 63.713.1 78.9 6.3 67.4 7.1 AD-1135998.1 45.7 6.7 42.2 3.3 59.4 18.4AD-1135999.1 39.8 6.5 61.4 10.2 67.1 14.9 AD-1136000.1 59.6 8.2 60.715.3 58.8 4.7 AD-1136001.1 29.4 7.6 42.2 11.7 47.3 3.1 AD-1136002.1 35.26.9 35.1 6.1 36.4 6.1 AD-1136003.1 59.4 9.1 55.3 8.4 51.2 7.4AD-1136004.1 39.8 4.4 59.7 4.9 62.2 12.8 AD-1136005.1 74.9 7.7 97.7 35.881.3 11.8 AD-1136006.1 53.9 7.3 87.2 11.7 68.6 11.2 AD-1136007.1 34.07.7 55.2 5.3 55.5 2.9 AD-1136008.1 26.6 9.2 46.9 22.0 55.4 9.7AD-1136009.1 41.9 11.4 69.5 25.5 65.4 10.1 AD-1136010.1 86.3 20.6 100.720.6 77.0 10.3 AD-1136011.1 84.9 11.0 91.8 14.5 63.6 6.0 AD-1136012.143.6 11.3 59.6 4.9 67.0 8.4 AD-1136013.1 74.1 8.8 96.5 30.1 68.6 13.1AD-1136014.1 82.1 16.3 83.9 13.5 68.5 6.9 AD-1136015.1 47.4 6.1 78.1 3.467.1 10.2 AD-1136016.1 45.3 19.8 69.7 10.5 63.3 5.7 AD-1136017.1 41.35.8 77.8 14.2 59.2 4.0 AD-1136018.1 47.4 10.9 64.5 11.1 64.4 10.8AD-1136019.1 85.6 9.7 97.4 9.3 67.8 2.9 AD-1136020.1 54.7 8.5 68.9 9.459.7 1.8 AD-1136021.1 40.9 16.6 60.7 5.1 71.3 24.0 AD-1136022.1 43.0 8.057.9 12.5 62.5 9.2 AD-1136023.1 46.1 20.5 71.9 8.1 60.6 4.8 AD-1136024.143.4 3.0 68.9 12.2 57.3 15.8 AD-1136025.1 61.1 9.2 74.0 6.2 69.6 13.1AD-1136026.1 108.6 14.0 107.0 13.2 75.9 11.1 AD-1136027.1 120.4 32.7104.1 23.0 77.3 11.7 AD-1136028.1 84.3 14.0 101.4 13.8 71.1 2.5AD-1136029.1 120.4 32.1 94.0 7.1 86.3 17.7 AD-1136030.1 37.1 4.1 48.14.1 56.4 4.2 AD-1136031.1 55.4 26.1 75.1 14.6 69.7 16.4 AD-1136032.133.4 7.4 47.8 10.1 38.4 3.3 AD-1136033.1 57.6 20.1 77.1 7.3 60.6 4.2AD-1136034.1 51.7 9.7 59.4 9.0 63.8 6.7 AD-1136035.1 58.2 10.8 89.8 10.864.4 3.0 AD-1136036.1 50.8 6.1 63.8 4.7 65.1 12.6 AD-1136037.1 41.8 7.750.4 3.1 64.3 17.4 AD-1136038.1 29.8 12.3 46.9 2.6 46.6 8.7 AD-1136039.128.7 11.8 37.0 5.6 64.7 29.0 AD-1136040.1 49.1 7.4 87.9 12.5 57.7 21.1AD-1136041.1 66.6 18.3 99.9 8.1 78.2 14.9 AD-1136042.1 45.1 15.3 72.35.2 77.1 17.8 AD-1136043.1 37.3 21.7 53.9 6.4 60.2 9.1 AD-1136044.1 74.824.4 106.8 8.2 76.4 8.0 AD-1136045.1 43.6 10.2 92.3 15.6 69.8 12.7AD-1136046.1 36.5 10.3 66.8 5.5 75.4 12.4 AD-1136047.1 40.5 10.3 71.09.0 67.4 19.4 AD-1136048.1 48.8 14.4 60.1 7.6 57.8 7.1 AD-1136049.1 82.519.0 91.0 17.0 63.6 6.6 AD-1136050.1 50.0 4.6 73.8 14.9 62.4 4.0AD-1136051.1 28.7 1.3 44.3 7.6 47.7 8.2 AD-1136052.1 27.2 7.6 55.7 11.449.6 3.7 AD-1136053.1 33.6 22.8 49.1 7.9 54.4 4.7 AD-1136054.1 41.4 21.261.3 6.3 55.8 6.9 AD-1136055.1 58.3 16.1 78.4 6.8 63.1 9.9 AD-1136056.150.5 13.4 76.7 10.5 65.5 11.3 AD-1136057.1 47.0 13.0 70.4 15.6 55.4 2.8AD-1136058.1 47.9 10.2 83.8 6.9 65.6 11.6 AD-1136059.1 76.2 14.1 95.417.7 67.3 11.0 AD-1136060.1 48.0 17.1 79.8 14.6 68.9 14.5 AD-1136061.128.0 5.2 53.9 10.6 53.8 13.6 AD-1136062.1 42.1 10.4 55.4 7.3 56.7 4.8AD-1136063.1 90.1 40.7 79.3 2.8 62.9 11.6 AD-1136064.1 58.7 14.6 77.93.2 72.4 16.2 AD-1136065.1 78.2 16.0 83.8 11.7 62.9 6.1 AD-1136066.150.9 17.0 74.0 7.0 64.3 13.4 AD-1136067.1 60.6 11.1 78.6 5.1 60.6 7.6AD-1136068.1 46.9 20.3 64.2 8.0 57.4 3.4 AD-1136069.1 33.5 10.4 34.810.9 54.5 4.7 AD-1136070.1 33.7 5.6 38.2 6.2 55.9 12.2 AD-1136071.1 43.77.9 47.8 5.6 82.7 10.9 AD-1136072.1 53.1 16.0 52.0 15.3 82.5 4.1AD-1136073.1 37.8 9.0 38.5 9.0 56.7 11.1 AD-1136074.1 59.0 7.7 56.7 13.953.4 14.8 AD-1136075.1 25.9 5.2 36.6 6.8 32.5 10.8 AD-1136076.1 38.011.5 40.0 15.3 42.6 9.8 AD-1136077.1 33.2 4.7 39.2 10.9 70.9 15.7AD-1136078.1 53.5 7.7 59.0 4.6 84.5 5.9 AD-1136079.1 52.0 18.0 63.3 13.191.1 12.3 AD-1136080.1 49.3 19.0 67.8 16.7 97.7 18.9 AD-1136081.1 29.05.4 40.0 13.8 63.7 12.3 AD-1136082.1 28.9 19.2 26.2 4.5 39.4 5.4AD-1136083.1 17.6 1.9 18.7 2.7 24.6 2.9 AD-1136084.1 27.4 3.7 32.3 7.843.4 8.6 AD-1136085.1 35.0 8.9 41.8 18.1 83.9 20.0 AD-1136086.1 74.423.0 78.3 11.0 102.6 14.3 AD-1136087.1 67.6 29.6 33.7 7.6 81.8 16.9AD-1136088.1 27.4 4.9 52.3 2.4 78.4 5.9 AD-1136089.1 37.2 18.0 33.6 1.761.0 15.7 AD-1136090.1 56.0 24.2 67.8 5.6 77.0 5.9 AD-1136091.1 23.2 4.332.6 13.8 44.2 7.7 AD-1136092.1 35.3 11.0 58.7 23.6 87.8 22.4AD-1136093.1 44.8 11.5 43.7 14.0 81.5 18.1 AD-1136094.1 47.6 12.0 47.89.7 94.1 28.6 AD-1136095.1 69.2 14.9 69.7 8.4 96.6 11.1 AD-1136096.167.0 21.6 73.8 5.2 94.4 4.7 AD-1136097.1 84.8 9.5 76.0 8.6 93.5 15.0AD-1136098.1 58.1 3.2 49.0 3.3 74.4 15.8 AD-1136099.1 63.7 17.1 72.721.6 112.8 14.8 AD-1136100.1 40.7 4.6 39.7 9.2 76.0 13.5 AD-1136101.149.5 11.9 39.8 3.4 67.2 2.7 AD-1136102.1 49.3 3.4 40.5 4.4 77.7 11.2AD-1136103.1 44.7 6.8 49.9 4.6 82.0 11.3 AD-1136104.1 56.9 13.7 52.9 5.487.6 5.3 AD-1136105.1 69.7 29.4 60.4 10.7 79.9 9.4 AD-1136106.1 28.3 8.436.9 7.8 54.4 9.8 AD-1136107.1 35.4 8.3 61.2 18.1 84.4 3.2 AD-1136108.149.1 20.7 49.7 12.8 75.4 17.4 AD-1136109.1 65.0 17.5 63.6 10.2 95.5 10.9AD-1136110.1 121.2 18.0 91.7 14.5 102.9 7.7 AD-1136111.1 85.6 9.1 80.817.2 96.8 12.5 AD-1136112.1 49.6 3.0 46.6 8.9 72.9 12.9 AD-1136114.145.2 9.2 37.7 18.3 71.0 4.6 AD-1136115.1 76.2 8.7 80.3 16.2 94.8 6.0AD-1136116.1 108.7 15.0 105.2 23.4 119.1 31.1 AD-1136117.1 45.8 8.8 37.14.1 56.7 6.1 AD-1136118.1 51.8 12.1 43.7 10.2 64.7 8.0 AD-1136119.1 68.79.8 53.1 7.7 83.8 6.9 AD-1136120.1 54.9 7.9 45.6 5.9 80.8 20.4AD-1136121.1 52.4 19.3 64.3 12.8 79.1 19.4 AD-1136122.1 103.8 15.0 91.113.4 113.7 22.7 AD-1136123.1 57.7 5.0 62.6 13.3 95.9 7.3 AD-1136124.169.7 5.7 74.6 5.3 96.5 21.5 AD-1136125.1 47.0 9.3 49.2 5.0 75.2 20.0AD-1136126.1 58.9 1.7 57.8 13.0 84.7 9.7 AD-1136127.1 50.2 11.1 33.2 5.158.2 5.2 AD-1136128.1 111.8 17.4 81.9 3.7 104.2 25.1 AD-1136129.1 77.634.6 100.1 45.8 79.2 12.9 AD-1136130.1 51.5 2.4 62.2 9.0 98.9 17.0AD-1136131.1 78.0 13.8 66.1 4.7 95.2 12.6 AD-1136132.1 73.5 9.3 68.8 3.1104.0 12.2 AD-1136133.1 65.9 4.8 66.8 16.8 89.7 9.7 AD-1136134.1 72.813.6 57.5 4.6 84.9 5.0 AD-1136135.1 57.0 17.8 59.3 9.0 86.4 6.9AD-1136136.1 49.1 25.8 58.5 4.6 63.5 7.5 AD-1136137.1 50.4 8.5 57.1 18.791.4 19.0 AD-1136138.1 83.8 21.2 72.2 13.5 92.9 7.0 AD-1136139.1 44.69.9 39.8 7.5 60.0 10.4 AD-1136140.1 75.1 8.6 64.3 2.4 89.1 11.8AD-1136141.1 36.6 3.5 30.3 2.9 61.7 13.8 AD-1136142.1 38.7 11.0 38.3 7.163.9 4.8 AD-1136143.1 34.9 13.9 46.9 16.2 66.0 23.0 AD-1136144.1 39.16.9 50.6 2.9 75.2 16.3 AD-1136145.1 58.8 3.7 55.3 7.0 84.5 19.3AD-1136146.1 69.4 4.6 67.7 12.3 93.8 12.6 AD-1136147.1 118.8 30.5 93.915.8 99.0 8.6 AD-1136148.1 65.9 17.7 62.0 4.9 82.9 3.2 AD-1136149.1 60.123.6 43.5 4.7 81.8 7.4 AD-1136150.1 30.2 6.2 43.5 12.6 86.1 10.5AD-1136151.1 47.3 10.5 70.6 23.5 66.4 9.4 AD-1136152.1 56.8 25.0 63.518.1 70.5 25.6 AD-1136153.1 52.3 8.3 49.4 13.5 77.7 7.8 AD-1136154.147.3 17.6 49.5 5.8 85.6 20.7 AD-1136155.1 106.6 1.5 76.3 15.7 92.6 6.2AD-1136156.1 36.6 9.6 50.8 32.7 65.3 8.2 AD-1136157.1 30.2 16.8 36.815.8 67.0 8.9 AD-1136158.1 54.9 10.5 68.4 15.9 78.3 18.4 AD-1136159.127.6 5.5 50.6 19.2 93.9 20.2 AD-1136160.1 31.7 5.8 53.7 16.2 121.0 34.1AD-1136161.1 32.0 5.4 62.0 25.2 68.9 9.8 AD-1136162.1 27.5 4.8 46.0 10.998.4 37.9 AD-1136163.1 21.6 7.4 33.8 6.6 52.8 26.7 AD-1136164.1 47.925.0 64.4 13.8 81.3 9.8 AD-1136165.1 31.9 6.9 62.1 2.0 56.8 11.0AD-1136166.1 20.5 3.9 35.5 6.5 52.0 26.5 AD-1136167.1 55.5 10.9 88.131.7 87.4 8.2 AD-1136168.1 53.0 3.7 64.5 18.7 91.7 31.7 AD-1136169.127.7 2.4 32.0 6.6 50.6 7.6 AD-1136170.1 28.0 2.5 39.1 16.3 61.7 6.3AD-1136171.1 78.1 6.6 54.6 21.1 96.7 4.1 AD-1136172.1 43.7 9.8 58.0 19.082.1 7.5 AD-1136173.1 34.8 6.7 62.4 10.9 78.4 9.5 AD-1136174.1 29.3 6.959.8 15.4 61.3 22.5 AD-1136175.1 39.0 6.3 57.8 26.0 70.6 16.2AD-1136176.1 71.2 7.3 64.8 22.4 107.7 18.8 AD-1136177.1 66.4 5.4 64.927.3 90.1 8.1 AD-1136178.1 47.5 8.5 61.3 17.3 87.4 8.7 AD-1136179.1 45.92.8 45.9 15.6 80.8 17.7 AD-1136180.1 56.6 18.0 59.3 7.1 66.9 5.1AD-1136181.1 28.8 5.9 41.2 4.2 35.8 3.4 AD-1136182.1 66.9 34.9 82.1 28.4129.7 48.8 AD-1136183.1 100.7 16.6 54.8 18.7 105.8 8.5 AD-1136184.1 72.69.2 51.5 8.0 113.2 16.5 AD-1136185.1 35.6 8.3 36.4 11.7 63.9 12.1AD-1136186.1 40.0 14.4 35.5 13.1 49.2 8.6 AD-1136187.1 28.6 2.8 29.711.3 34.5 9.3 AD-1136188.1 42.9 6.3 53.5 11.6 74.4 49.5 AD-1136189.142.1 7.5 27.5 6.4 82.1 4.1 AD-1136190.1 52.0 21.2 51.7 20.4 102.1 6.2AD-1136191.1 34.8 5.6 42.0 17.6 51.1 10.0 AD-1136192.1 53.3 7.0 47.912.6 62.1 3.1 AD-1136193.1 44.7 6.5 46.9 5.6 44.0 8.3 AD-1136194.1 41.45.9 58.6 17.5 60.4 16.8 AD-1136195.1 55.5 6.3 47.9 19.4 58.2 14.3AD-1136196.1 37.3 4.0 45.8 11.9 47.8 11.2 AD-1136197.1 39.2 7.4 40.012.2 71.4 25.0 AD-1136198.1 68.5 16.0 78.5 25.7 91.1 11.8 AD-1136199.155.3 5.4 93.1 18.1 71.0 21.0 AD-1136200.1 70.6 4.0 89.1 9.9 75.5 23.2AD-1136201.1 57.1 10.4 76.0 12.2 88.0 13.0 AD-1136202.1 69.9 15.3 68.66.4 53.2 11.9 AD-1136203.1 57.4 8.3 103.7 29.4 44.9 4.9 AD-1136204.138.2 7.2 55.3 8.9 104.7 41.6 AD-1136205.1 47.9 5.5 88.6 26.9 111.5 23.5AD-1136206.1 81.7 13.0 109.4 16.9 107.2 13.7 AD-1136207.1 68.2 7.2 101.310.3 78.6 14.3 AD-1136208.1 71.8 4.9 102.4 18.6 76.5 33.1 AD-1136209.174.7 10.1 100.9 37.2 65.8 17.0 AD-1136210.1 56.3 2.9 73.5 19.8 57.7 10.7AD-1136211.1 65.2 7.3 100.2 28.2 56.4 4.2 AD-1136212.1 68.0 9.0 80.427.1 95.4 30.6 AD-1136213.1 61.3 11.2 83.6 7.5 87.1 11.6 AD-1136214.142.3 7.1 64.9 10.9 67.7 12.0 AD-1136215.1 52.8 3.6 65.0 6.1 55.1 16.4AD-1136216.1 69.3 8.4 97.1 12.4 77.4 15.7 AD-1136217.1 53.8 6.9 87.422.9 81.9 20.1 AD-1136218.1 54.8 7.2 71.0 21.4 66.1 9.4 AD-1136219.146.9 10.2 67.7 26.7 44.6 4.5 AD-1136220.1 53.5 18.0 53.3 17.1 71.6 48.4AD-1136221.1 33.1 2.5 61.5 2.1 82.6 28.7 AD-1136222.1 79.1 6.2 88.6 8.365.8 16.5 AD-1136223.1 47.6 1.8 68.0 6.1 58.6 12.7 AD-1136224.1 40.2 4.463.3 7.5 50.3 16.6 AD-1136225.1 29.8 4.6 53.7 10.7 61.3 44.7AD-1136226.1 46.1 5.2 68.0 23.6 63.3 23.9 AD-1136227.1 40.9 3.9 63.010.2 73.8 19.3 AD-1136228.1 51.9 4.9 96.7 7.6 87.7 22.9 AD-1136229.144.5 4.0 74.2 3.7 72.2 22.1 AD-1136230.1 47.2 10.2 59.5 3.9 61.6 14.2AD-1136231.1 41.2 4.9 61.0 13.5 59.6 20.1 AD-1136232.1 38.2 3.0 54.910.1 47.9 7.5 AD-1136233.1 37.2 3.9 63.7 12.1 52.9 12.6 AD-1136234.141.4 6.6 48.2 6.9 81.9 30.6 AD-1136235.1 87.4 19.0 80.6 22.0 119.7 40.0AD-1136236.1 64.4 12.3 81.2 7.2 85.9 35.2 AD-1136237.1 80.5 4.3 125.818.4 97.4 21.2 AD-1136238.1 72.1 9.4 85.2 13.8 136.5 56.4 AD-1136239.163.0 18.5 96.0 28.1 103.5 37.2 AD-1136240.1 45.1 5.4 70.7 10.4 54.5 15.1AD-1136241.1 56.8 3.7 72.3 32.0 56.3 14.8 AD-1136242.1 40.6 11.9 63.128.3 88.7 39.4 AD-1136243.1 30.8 4.4 51.7 10.8 65.4 29.0 AD-1136244.163.3 2.2 90.1 36.7 95.2 36.8 AD-1136245.1 94.6 13.8 91.9 26.1 116.1 27.6AD-1136246.1 70.0 3.9 76.0 20.1 73.2 13.3 AD-1136247.1 45.3 7.2 46.716.3 70.7 51.5 AD-1136248.1 63.1 4.0 73.3 19.8 57.4 23.2 AD-1136249.159.2 14.4 59.4 6.1 81.5 11.1 AD-1136250.1 64.4 13.2 67.4 9.5 95.8 4.8AD-1136251.1 87.5 17.7 63.5 11.6 97.1 17.0 AD-1136252.1 60.1 7.2 63.57.9 88.6 27.3 AD-1136253.1 75.8 10.0 84.2 13.3 95.7 37.6 AD-1136254.164.6 11.0 59.6 7.9 91.7 9.5 AD-1136255.1 56.8 7.8 70.0 22.1 97.7 19.5AD-1136256.1 24.7 2.9 38.0 4.3 88.3 24.6 AD-1136257.1 54.9 10.8 46.2 6.868.8 7.2 AD-1136258.1 71.1 4.1 64.2 8.4 95.1 11.8 AD-1136259.1 85.2 13.469.2 3.5 106.0 23.0 AD-1136260.1 59.0 9.7 60.9 7.5 95.2 21.8AD-1136261.1 70.1 19.7 63.3 1.9 122.1 31.3 AD-1136262.1 68.1 9.8 67.64.5 105.6 11.9 AD-1136263.1 39.0 9.5 46.6 8.0 97.1 20.0 AD-1136264.126.3 2.8 37.6 9.3 72.9 19.0 AD-1136265.1 53.2 6.5 50.2 7.8 77.0 8.4AD-1136266.1 79.7 14.9 65.4 9.1 99.3 7.4 AD-1136267.1 100.0 13.4 81.84.3 98.6 9.9 AD-1136268.1 54.4 4.0 55.7 7.5 91.8 23.7 AD-1136269.1 32.08.1 40.6 8.0 90.8 20.1 AD-1136270.1 37.5 10.0 50.7 16.5 81.7 19.1AD-1136271.1 32.3 8.5 42.5 8.3 60.6 11.8 AD-1136272.1 57.7 6.4 67.2 9.978.6 6.4 AD-1136273.1 87.8 21.5 95.4 16.5 103.5 17.7 AD-1136274.1 80.19.4 69.6 7.3 119.3 16.5 AD-1136275.1 71.4 12.2 76.8 14.1 139.8 50.5AD-1136276.1 61.3 18.8 63.9 11.2 91.0 15.3 AD-1136277.1 41.7 2.0 43.610.3 92.3 11.5 AD-1136278.1 30.2 5.0 45.4 5.2 77.8 6.3 AD-1136279.1 52.30.2 49.2 9.6 80.5 12.8 AD-1136280.1 65.8 6.6 62.5 5.6 90.5 16.8AD-1136281.1 70.3 4.6 68.3 3.8 124.4 52.7 AD-1136282.1 64.0 9.5 55.3 5.994.2 2.1 AD-1136283.1 58.9 14.2 62.4 6.3 103.8 7.2 AD-1136284.1 53.3 7.079.0 14.2 148.3 31.4 AD-1136285.1 48.7 4.5 53.6 7.0 93.2 15.7AD-1136286.1 29.4 1.9 50.9 11.8 58.6 6.9 AD-1136287.1 57.6 14.7 52.6 6.263.6 9.9 AD-1136288.1 96.4 35.9 74.5 7.6 98.4 13.6 AD-1136289.1 66.210.5 69.8 12.1 110.1 12.8 AD-1136290.1 59.7 19.2 54.8 3.9 108.6 10.5AD-1136291.1 57.5 11.2 56.6 8.5 85.8 5.4 AD-1136292.1 57.3 6.2 59.8 6.7109.5 9.9 AD-1136293.1 66.8 6.3 55.7 7.6 90.5 24.6 AD-1136294.1 86.811.5 68.2 4.1 103.1 15.7 AD-1136295.1 83.1 6.9 84.9 6.3 103.9 18.6AD-1136296.1 90.8 8.5 67.7 5.9 124.8 20.9 AD-1136297.1 86.0 13.8 91.816.6 132.2 29.3 AD-1136298.1 116.6 18.2 73.3 8.6 140.8 57.1 AD-1136299.184.6 12.3 70.7 14.7 100.7 27.4 AD-1136300.1 78.1 12.2 73.0 17.7 111.827.6 AD-1136301.1 58.9 3.1 64.4 7.4 76.3 6.0 AD-1136302.1 72.2 12.6 76.216.9 111.1 24.3 AD-1136303.1 91.1 11.6 99.4 17.6 130.4 26.5 AD-1136304.1113.3 17.8 90.2 16.8 129.1 22.4 AD-1136305.1 129.1 31.2 98.9 7.3 132.219.6 AD-1136306.1 114.5 12.4 91.2 5.5 115.6 8.2 AD-1136307.1 102.8 22.091.1 9.2 132.4 22.8 AD-1136308.1 53.3 7.5 55.0 7.1 65.8 12.9AD-1136309.1 31.8 3.9 51.1 4.8 55.0 13.3 AD-1136310.1 77.5 10.6 55.6 8.879.3 14.8 AD-1136311.1 79.4 13.0 73.2 4.2 95.4 19.9 AD-1136312.1 93.19.0 75.6 15.4 102.9 20.8 AD-1136313.1 63.2 5.1 53.8 4.0 103.8 33.1AD-1136314.1 62.9 7.5 54.4 7.4 67.1 13.3 AD-1136315.1 60.1 7.5 59.3 10.290.7 46.5 AD-1136316.1 39.6 8.1 56.6 9.3 81.4 8.6 AD-1136317.1 77.6 28.155.9 9.6 61.6 10.0 AD-1136318.1 73.8 12.7 58.3 4.1 98.9 37.1AD-1136319.1 76.7 7.6 61.5 2.8 112.7 15.4 AD-1136320.1 80.1 23.9 61.26.0 109.4 18.5 AD-1136321.1 56.6 3.5 45.1 10.5 72.2 8.3 AD-1136322.163.0 11.0 44.2 11.9 71.4 9.7 AD-1136323.1 55.4 6.9 58.5 9.1 82.9 33.3AD-1136324.1 53.8 12.8 55.5 17.5 59.5 18.1 AD-1136325.1 71.9 2.9 70.49.6 70.8 14.0 AD-1136326.1 64.3 5.6 59.0 11.8 63.3 18.4 AD-1136327.1124.1 12.1 78.0 8.7 82.4 18.6 AD-1136328.1 68.2 15.9 57.0 7.0 74.3 2.4AD-1136329.1 63.1 1.4 42.8 4.8 65.1 17.1 AD-1136330.1 81.2 11.6 58.315.0 59.9 8.6 AD-1136331.1 67.7 12.3 56.1 17.5 48.3 2.3 AD-1136332.177.2 11.5 61.1 8.7 60.2 43.9 AD-1136333.1 67.6 8.3 46.4 9.6 63.5 13.5AD-1136334.1 54.7 4.7 47.0 6.7 49.5 19.6 AD-1136335.1 70.7 23.3 55.9 7.372.2 6.0 AD-1136336.1 61.4 16.0 47.3 9.7 48.6 3.0 AD-1136337.1 68.7 11.858.5 16.7 57.7 8.7 AD-1136338.1 78.3 18.3 57.4 13.1 64.1 15.3

TABLE 5 Xanthine Dehydrogenase dsRNA Agent In Vitro Single Dose Screensin Primary Cynomolgus Hepatocytes PCH 500 nM 100 nM 10 nM 0.1 nM % ofAvg % of Avg % of Avg % of Avg Message ST Message ST Message ST MessageST Duplex ID Remaining DEV Remaining DEV Remaining DEV Remaining DEVAD-1135979.1 138.0 20.2 110.0 11.0 94.0 82.2 113.3 10.0 AD-1135980.160.3 11.1 124.1 15.0 152.1 57.1 124.6 23.3 AD-1135981.1 115.1 33.6 112.619.9 173.7 5.1 132.5 6.6 AD-1135982.1 100.6 20.8 140.8 25.5 119.8 74.8146.2 17.9 AD-1135983.1 103.9 29.8 138.0 20.5 181.2 36.9 116.5 27.6AD-1135984.1 104.4 31.9 138.2 33.1 93.9 74.7 105.6 4.1 AD-1135985.1 72.30.4 149.0 11.5 131.7 32.2 92.2 9.0 AD-1135986.1 101.6 34.7 122.8 34.9160.1 42.9 107.8 2.1 AD-1135987.1 77.8 7.0 99.5 37.7 102.4 24.9 96.8 1.4AD-1135988.1 107.4 22.1 141.2 40.1 118.1 22.1 103.6 7.9 AD-1135989.176.6 28.0 154.9 44.7 134.2 15.6 117.2 30.5 AD-1135990.1 110.7 26.7 115.432.1 172.5 27.7 118.0 18.5 AD-1135991.1 89.0 18.5 117.4 40.9 118.0 37.5109.0 28.3 AD-1135992.1 82.2 22.3 132.5 15.0 131.4 17.8 113.9 8.5AD-1135993.1 84.9 17.3 137.0 22.9 107.0 18.4 112.8 6.2 AD-1135994.1 94.13.2 101.9 23.4 124.7 25.3 109.1 24.7 AD-1135995.1 93.4 21.3 111.5 28.2120.0 14.0 87.7 12.5 AD-1135996.1 85.0 6.0 157.7 64.6 142.4 17.5 134.130.0 AD-1135997.1 67.4 6.9 117.1 35.1 128.1 33.4 125.0 20.4 AD-1135998.177.2 11.3 131.9 62.5 99.5 27.0 111.0 19.9 AD-1135999.1 75.6 11.2 94.34.3 102.1 20.1 103.1 23.0 AD-1136000.1 82.0 11.1 125.5 37.5 106.1 13.5118.5 14.2 AD-1136001.1 59.7 5.8 103.7 32.6 94.7 38.1 110.8 2.2AD-1136002.1 60.2 4.1 92.6 20.9 127.4 14.7 96.5 14.6 AD-1136003.1 66.18.7 102.6 31.8 116.8 15.5 124.7 18.1 AD-1136004.1 77.0 18.8 85.8 32.3134.9 22.7 140.4 22.5 AD-1136005.1 88.5 10.2 86.9 41.1 113.1 28.5 101.329.5 AD-1136006.1 83.2 2.1 99.4 40.8 89.2 11.1 95.8 7.1 AD-1136007.156.8 16.1 116.8 44.8 121.3 23.8 103.6 13.2 AD-1136008.1 79.6 17.1 93.427.4 155.3 32.6 81.5 25.5 AD-1136009.1 65.8 4.8 95.4 34.1 156.8 25.5101.6 15.2 AD-1136010.1 87.1 18.6 81.0 24.9 94.1 34.9 109.8 12.7AD-1136011.1 82.3 5.7 93.9 40.1 94.0 19.6 130.1 19.9 AD-1136012.1 71.514.2 114.2 12.5 116.8 38.3 124.7 9.9 AD-1136013.1 68.9 8.8 129.5 46.191.6 6.2 119.2 9.8 AD-1136014.1 73.4 10.5 68.6 10.6 106.6 56.2 104.1 7.0AD-1136015.1 71.8 4.9 90.5 16.0 98.2 41.2 101.7 16.1 AD-1136016.1 78.65.7 82.7 10.9 130.1 140.2 120.4 4.7 AD-1136017.1 59.2 6.3 83.5 24.7 82.356.2 81.9 1.2 AD-1136018.1 57.7 12.0 64.2 19.9 105.4 11.3 98.8 22.8AD-1136019.1 67.2 10.5 93.1 25.7 102.7 15.4 107.9 10.1 AD-1136020.1 75.419.4 87.3 38.5 101.6 22.5 126.2 13.2 AD-1136021.1 61.3 3.6 65.8 6.1 77.114.4 113.4 17.8 AD-1136022.1 59.2 22.6 75.0 13.3 116.4 48.4 107.3 17.4AD-1136023.1 71.9 21.4 76.1 14.2 100.8 38.3 91.6 16.3 AD-1136024.1 82.18.1 81.4 35.1 116.6 5.3 83.7 4.5 AD-1136025.1 75.8 30.1 107.4 32.7 115.55.3 87.4 15.6 AD-1136026.1 69.2 11.6 127.8 46.6 97.5 24.8 95.5 18.1AD-1136027.1 86.9 8.3 118.5 19.4 124.4 28.7 116.4 24.3 AD-1136028.1 84.230.1 139.7 31.6 95.7 7.5 125.4 18.6 AD-1136029.1 75.1 8.4 82.5 19.0 74.38.3 93.3 12.2 AD-1136030.1 44.5 5.6 84.2 33.7 120.3 51.2 99.0 14.8AD-1136031.1 66.2 9.8 96.0 46.4 117.0 67.0 80.8 16.5 AD-1136032.1 62.116.0 65.9 13.8 91.3 60.9 82.6 11.3 AD-1136033.1 61.5 7.8 83.1 16.5 123.623.4 102.5 17.5 AD-1136034.1 54.3 16.6 80.6 13.2 101.9 7.7 105.9 6.3AD-1136035.1 78.6 19.3 93.1 20.7 112.6 15.2 117.2 4.8 AD-1136036.1 79.45.2 77.1 19.8 159.8 86.1 105.2 23.1 AD-1136037.1 59.5 16.2 89.9 32.771.5 11.2 100.6 3.3 AD-1136038.1 56.9 11.1 68.5 10.1 98.6 41.5 95.9 15.9AD-1136039.1 49.4 7.2 61.8 25.1 153.5 77.6 74.7 13.8 AD-1136040.1 52.113.1 84.6 35.1 155.3 9.7 72.5 7.5 AD-1136041.1 71.9 17.8 75.0 29.6 89.410.9 91.1 9.1 AD-1136042.1 85.2 25.0 76.0 6.6 120.5 23.3 113.6 14.8AD-1136043.1 89.3 21.7 89.1 45.3 110.0 4.8 92.9 23.7 AD-1136044.1 81.616.4 66.5 15.2 101.0 38.9 92.7 23.4 AD-1136045.1 68.2 3.6 75.1 13.4 84.422.8 92.8 12.6 AD-1136046.1 75.8 7.4 90.3 25.3 71.8 12.2 71.5 3.7AD-1136047.1 94.8 5.1 98.7 40.9 134.8 25.3 70.6 2.4 AD-1136048.1 78.618.3 84.4 22.1 92.4 15.9 71.7 4.1 AD-1136049.1 91.4 32.0 84.3 19.5 111.317.6 113.8 13.0 AD-1136050.1 64.1 15.1 89.4 27.9 101.3 45.3 97.5 23.8AD-1136051.1 44.3 7.4 67.5 28.3 86.1 18.7 107.4 25.9 AD-1136052.1 62.317.5 69.8 32.2 86.2 19.2 77.4 21.0 AD-1136053.1 57.0 5.6 77.8 20.7 131.435.3 68.9 22.1 AD-1136054.1 59.2 17.7 75.6 21.8 97.2 26.8 80.9 6.7AD-1136055.1 74.7 13.5 94.8 20.5 74.2 19.1 88.6 11.4 AD-1136056.1 70.819.5 85.1 20.8 117.7 26.0 92.1 4.1 AD-1136057.1 78.2 3.3 125.3 24.0127.3 31.9 93.4 12.6 AD-1136058.1 73.7 15.3 64.5 13.5 113.2 21.3 81.723.1 AD-1136059.1 84.5 9.5 90.3 28.2 118.2 61.6 81.6 15.8 AD-1136060.172.5 14.4 70.5 22.8 80.7 13.0 65.1 14.1 AD-1136061.1 60.8 20.7 84.8 15.899.7 27.0 54.9 1.6 AD-1136062.1 96.7 25.3 60.3 2.9 75.1 18.2 56.1 29.9AD-1136063.1 87.6 7.4 63.4 14.6 72.7 15.4 69.6 9.6 AD-1136064.1 77.711.2 91.6 7.0 152.9 110.5 99.5 32.0 AD-1136065.1 64.0 13.4 80.7 20.681.6 22.8 72.8 5.9 AD-1136066.1 45.0 9.3 93.1 22.7 91.3 24.0 74.5 11.0AD-1136067.1 82.1 15.8 71.3 9.4 111.2 40.5 65.7 7.6 AD-1136068.1 59.57.1 65.8 7.8 46.3 3.4 59.5 9.9 AD-1136069.1 70.5 11.2 110.1 34.4 93.59.2 116.6 15.6 AD-1136070.1 72.7 14.9 97.8 10.1 123.2 9.0 119.7 19.3AD-1136071.1 89.3 29.7 147.2 32.6 116.4 9.6 120.2 22.4 AD-1136072.1 95.510.2 119.5 39.2 126.8 29.4 122.5 5.6 AD-1136073.1 87.5 9.9 109.6 20.4129.3 13.0 129.0 17.8 AD-1136074.1 79.1 16.2 120.6 3.2 109.1 10.5 130.813.1 AD-1136075.1 82.2 18.7 96.5 22.5 105.9 9.6 126.9 18.4 AD-1136076.191.2 18.0 85.6 27.6 85.6 12.4 110.3 23.2 AD-1136077.1 74.5 16.0 99.010.9 104.5 14.7 104.4 13.8 AD-1136078.1 100.1 9.6 130.6 11.9 113.5 15.7135.4 21.0 AD-1136079.1 101.0 14.8 143.2 10.1 127.3 15.3 140.5 24.4AD-1136080.1 96.1 19.2 146.9 13.4 135.5 16.3 117.8 19.4 AD-1136081.183.7 28.2 82.3 18.0 115.2 19.9 106.3 10.7 AD-1136082.1 72.0 11.5 97.019.0 94.7 23.0 101.9 10.1 AD-1136083.1 69.3 10.0 115.8 28.1 101.8 10.0113.2 9.7 AD-1136084.1 81.1 10.4 94.1 15.0 85.7 4.8 120.1 29.3AD-1136085.1 85.9 15.2 118.5 28.7 96.3 12.8 107.9 6.7 AD-1136086.1 83.119.4 129.9 14.1 141.3 1.7 119.5 25.6 AD-1136087.1 100.3 8.6 137.9 31.5118.5 23.2 105.9 21.7 AD-1136088.1 97.3 20.8 140.1 6.0 113.2 25.0 95.97.5 AD-1136089.1 98.5 16.6 121.8 27.9 101.3 13.0 94.3 31.5 AD-1136090.1106.2 20.8 140.4 15.5 101.2 11.2 99.1 9.0 AD-1136091.1 73.4 7.7 96.117.9 91.4 11.8 107.2 18.0 AD-1136092.1 72.0 15.5 107.6 7.0 94.3 10.6100.8 19.5 AD-1136093.1 85.7 19.5 105.7 25.1 126.6 14.6 122.6 27.1AD-1136094.1 82.4 14.4 141.4 17.8 128.2 4.1 143.9 43.9 AD-1136095.1 85.016.7 112.5 26.9 118.6 13.7 97.8 25.1 AD-1136096.1 104.5 14.0 119.3 28.0113.7 15.1 111.6 8.6 AD-1136097.1 94.4 18.7 121.0 28.7 99.6 22.6 81.07.2 AD-1136098.1 66.1 19.4 100.5 41.4 79.1 15.3 70.6 7.3 AD-1136099.168.3 4.4 88.1 9.5 111.4 7.6 105.9 10.8 AD-1136100.1 64.9 19.9 99.2 3.7112.4 17.2 123.6 20.1 AD-1136101.1 67.7 12.8 107.3 26.3 121.3 16.0 117.718.2 AD-1136102.1 86.3 24.3 132.6 11.5 138.2 14.7 144.2 15.2AD-1136103.1 81.7 18.6 121.1 32.7 100.0 19.9 124.6 27.2 AD-1136104.169.6 6.9 101.7 5.5 106.3 12.3 99.0 14.6 AD-1136105.1 83.9 26.6 96.0 28.488.3 6.1 95.9 16.0 AD-1136106.1 74.4 18.2 121.1 39.6 73.2 10.2 97.2 13.7AD-1136107.1 69.3 9.2 102.7 12.1 78.9 11.3 74.4 3.5 AD-1136108.1 69.59.8 105.9 9.3 96.8 19.0 111.2 8.6 AD-1136109.1 72.8 11.1 111.8 12.9114.2 8.7 122.4 20.9 AD-1136110.1 100.8 9.3 128.7 27.3 125.8 17.6 124.717.2 AD-1136111.1 94.8 10.0 95.0 18.0 104.9 15.7 100.7 15.9 AD-1136112.182.1 10.6 113.0 19.4 84.3 17.3 114.3 18.2 AD-1136114.1 56.6 8.9 101.721.4 90.1 5.0 98.0 36.5 AD-1136115.1 72.9 15.9 121.0 29.4 116.4 21.195.7 12.5 AD-1136116.1 82.9 5.1 110.2 7.2 100.3 8.4 88.8 11.4AD-1136117.1 63.2 26.4 97.7 4.8 119.2 27.4 90.4 13.0 AD-1136118.1 61.64.3 95.5 25.6 84.9 10.6 86.1 11.7 AD-1136119.1 82.1 15.1 116.6 5.7 93.117.2 95.9 8.6 AD-1136120.1 74.9 9.7 93.3 18.1 77.3 10.4 92.8 11.7AD-1136121.1 83.0 20.0 69.1 21.8 65.5 6.7 73.5 8.7 AD-1136122.1 68.410.0 90.0 11.9 97.5 12.8 90.8 8.0 AD-1136123.1 76.3 14.8 109.2 6.3 108.219.9 85.4 25.2 AD-1136124.1 76.1 9.4 97.8 18.2 98.9 14.5 96.9 16.9AD-1136125.1 77.7 21.2 100.9 17.9 115.5 27.9 109.8 18.2 AD-1136126.191.7 16.1 92.7 13.9 104.3 27.1 101.7 11.3 AD-1136127.1 58.6 8.7 82.424.5 88.4 17.5 83.0 11.3 AD-1136128.1 76.2 22.3 99.3 18.3 92.0 9.4 81.313.0 AD-1136129.1 68.2 14.1 68.7 22.7 71.5 17.4 66.4 15.3 AD-1136130.180.8 13.1 97.9 6.2 97.0 11.2 76.9 7.8 AD-1136131.1 76.6 19.3 113.4 2.7103.2 26.0 89.0 8.9 AD-1136132.1 78.2 9.9 123.5 16.4 132.2 21.3 110.419.0 AD-1136133.1 84.8 19.3 87.9 18.4 108.8 23.8 101.2 7.4 AD-1136134.179.4 5.0 94.6 5.1 96.5 15.0 110.6 21.3 AD-1136135.1 87.2 14.7 75.4 25.090.2 11.1 79.6 25.2 AD-1136136.1 65.1 6.8 55.1 31.4 69.7 12.2 63.4 15.5AD-1136137.1 83.6 9.9 107.0 0.5 102.7 17.4 96.2 6.1 AD-1136138.1 59.17.9 116.3 22.9 92.4 6.3 110.3 12.6 AD-1136139.1 80.1 7.5 102.2 15.4123.0 15.0 113.0 26.8 AD-1136140.1 77.9 15.8 73.4 17.7 107.6 12.2 103.524.3 AD-1136141.1 63.5 7.4 73.2 15.7 78.5 10.8 96.1 16.1 AD-1136142.172.4 15.8 63.2 11.8 76.8 14.6 73.9 22.0 AD-1136143.1 69.1 6.8 63.6 11.968.0 17.8 64.6 9.4 AD-1136144.1 80.1 8.4 75.1 10.5 99.9 19.3 83.1 4.2AD-1136145.1 83.3 5.0 97.0 21.2 96.8 9.8 97.4 19.6 AD-1136146.1 94.116.7 104.8 32.0 95.2 23.4 97.1 15.9 AD-1136147.1 97.9 8.6 112.2 15.3114.9 7.5 100.2 10.3 AD-1136148.1 86.8 11.6 72.8 24.6 95.8 12.9 100.120.1 AD-1136149.1 81.9 12.9 85.2 10.2 90.9 17.0 97.5 34.3 AD-1136150.171.1 5.3 80.7 16.4 87.1 12.8 94.7 34.4 AD-1136151.1 89.7 19.7 63.0 38.667.7 3.9 62.1 22.2 AD-1136152.1 84.8 17.6 93.9 21.0 105.4 14.2 67.3 8.3AD-1136153.1 80.9 9.8 105.6 2.5 101.6 8.9 87.8 12.2 AD-1136154.1 86.47.8 83.5 15.1 103.4 9.4 97.1 10.3 AD-1136155.1 90.9 23.6 112.1 16.9109.5 7.9 88.2 6.4 AD-1136156.1 69.2 12.9 82.9 26.6 93.4 9.8 88.3 12.9AD-1136157.1 76.5 9.5 75.5 16.4 96.3 12.5 82.6 10.7 AD-1136158.1 78.110.3 85.5 6.5 88.7 7.4 71.2 9.2 AD-1136159.1 43.3 40.2 121.4 41.6 109.511.3 143.7 73.3 AD-1136160.1 95.8 25.4 118.1 53.7 115.4 13.3 130.2 33.1AD-1136161.1 156.0 58.2 158.0 40.7 114.7 14.2 132.5 23.9 AD-1136162.169.3 23.9 120.9 33.9 119.8 12.2 160.5 24.0 AD-1136163.1 74.1 4.3 94.915.8 101.1 19.6 107.3 18.3 AD-1136164.1 102.0 13.5 162.8 81.5 97.7 33.3128.5 10.1 AD-1136165.1 78.9 28.8 144.5 32.9 92.1 21.7 134.9 48.1AD-1136166.1 75.5 34.6 129.8 26.6 75.9 136.2 54.8 AD-1136167.1 168.235.6 110.6 17.3 98.8 47.9 77.2 42.5 AD-1136168.1 107.5 25.5 98.5 14.6132.1 8.3 124.0 24.5 AD-1136169.1 77.7 14.0 93.4 13.3 87.9 24.6 99.9 8.8AD-1136170.1 87.2 23.0 113.2 28.8 115.9 4.3 145.8 19.7 AD-1136171.1 88.828.1 141.1 2.8 125.2 33.6 140.4 6.1 AD-1136172.1 101.1 18.3 106.3 21.5106.1 29.3 135.0 23.7 AD-1136173.1 89.1 26.0 105.4 20.8 96.5 17.9 88.910.9 AD-1136174.1 98.9 17.1 56.1 33.7 94.9 17.2 122.7 64.3 AD-1136175.1119.8 18.5 130.4 31.7 73.5 23.3 129.4 10.1 AD-1136176.1 95.1 17.7 132.310.0 128.0 17.0 125.7 17.9 AD-1136177.1 111.2 12.0 129.7 18.6 130.1 30.2109.7 6.2 AD-1136178.1 81.9 12.8 111.7 15.8 120.8 18.5 124.3 49.9AD-1136179.1 103.0 13.1 123.6 10.8 129.2 12.7 94.3 10.2 AD-1136180.194.1 14.4 119.8 20.6 109.1 8.7 104.5 5.6 AD-1136181.1 80.4 8.6 61.1 4.8103.7 32.6 98.9 20.2 AD-1136182.1 146.5 24.7 113.3 14.7 124.3 60.8 117.53.7 AD-1136183.1 81.8 16.0 114.8 23.7 130.1 19.2 129.3 19.0 AD-1136184.1100.5 10.5 140.0 26.6 119.3 27.8 109.2 18.4 AD-1136185.1 86.3 13.5 113.620.7 137.7 7.9 129.4 27.5 AD-1136186.1 68.2 10.7 114.0 3.0 130.5 24.9104.8 11.0 AD-1136187.1 85.9 20.0 97.3 17.9 103.1 18.0 91.6 4.3AD-1136188.1 87.2 6.2 126.8 6.8 103.7 17.8 95.3 25.2 AD-1136189.1 87.134.4 138.2 21.0 126.3 18.3 122.3 27.9 AD-1136190.1 116.7 40.3 121.6 10.3134.5 15.8 163.3 48.0 AD-1136191.1 105.2 15.4 130.1 34.0 124.4 16.8121.0 27.5 AD-1136192.1 97.3 17.1 151.8 11.8 131.1 30.0 145.5 16.0AD-1136193.1 93.6 21.5 110.8 20.2 130.3 34.5 116.7 25.5 AD-1136194.191.0 17.6 125.2 22.1 126.9 7.4 103.6 23.3 AD-1136195.1 109.6 21.9 92.919.4 108.0 13.5 81.9 12.5 AD-1136196.1 85.2 33.9 85.2 27.3 104.2 20.9100.2 10.9 AD-1136197.1 104.7 12.5 86.5 17.1 127.6 39.6 132.2 40.1AD-1136198.1 122.2 24.1 103.0 19.9 138.2 18.1 124.0 34.7 AD-1136199.176.7 17.3 95.9 7.4 116.7 12.3 111.4 12.1 AD-1136200.1 91.6 16.2 123.125.2 126.9 32.2 104.0 36.0 AD-1136201.1 73.6 15.4 99.6 26.1 83.8 11.371.5 19.1 AD-1136202.1 72.7 8.6 95.9 24.1 114.6 28.7 87.1 17.2AD-1136203.1 69.9 11.0 74.6 18.4 126.8 20.6 85.4 20.3 AD-1136204.1 99.953.1 89.2 16.7 137.2 4.5 138.9 4.8 AD-1136205.1 73.4 17.8 107.7 17.5144.7 21.8 136.0 38.7 AD-1136206.1 87.3 18.9 120.5 11.0 107.3 20.5 115.540.3 AD-1136207.1 85.5 21.6 112.6 26.2 81.9 28.3 74.5 4.6 AD-1136208.183.6 5.3 115.1 15.6 79.9 26.1 80.1 7.7 AD-1136209.1 74.1 16.9 83.9 7.0101.2 33.8 68.2 18.6 AD-1136210.1 79.2 15.2 92.9 17.0 92.2 25.5 80.112.7 AD-1136211.1 77.9 18.3 75.0 8.5 110.5 6.5 80.6 6.9 AD-1136212.176.2 41.5 109.6 24.4 118.0 16.6 139.1 3.9 AD-1136213.1 95.4 20.3 92.911.9 123.4 16.0 109.1 16.9 AD-1136214.1 62.4 14.1 76.6 6.7 89.2 17.3102.8 27.1 AD-1136215.1 63.4 16.7 105.3 15.1 100.4 34.7 73.6 25.3AD-1136216.1 66.8 17.6 82.2 29.4 127.5 22.5 108.9 16.2 AD-1136217.1 60.320.1 63.7 10.4 64.0 13.9 61.2 27.6 AD-1136218.1 66.6 13.3 60.5 8.7 70.114.6 65.6 16.3 AD-1136219.1 65.6 9.5 107.3 32.2 95.2 19.9 75.7 3.7AD-1136220.1 111.3 22.6 129.8 14.7 135.3 32.2 155.5 85.4 AD-1136221.185.1 10.8 101.0 8.3 102.0 23.9 105.4 25.9 AD-1136222.1 85.7 2.2 108.613.3 74.7 29.6 60.8 13.5 AD-1136223.1 76.1 23.3 65.4 13.2 76.0 30.7 79.317.7 AD-1136224.1 66.5 6.4 62.6 14.6 47.6 6.1 48.1 11.1 AD-1136225.167.1 7.3 51.4 20.4 48.0 11.6 55.6 13.0 AD-1136226.1 59.5 6.6 68.7 10.496.1 19.1 78.7 6.3 AD-1136227.1 107.2 4.1 125.4 21.5 80.0 13.7 112.274.8 AD-1136228.1 87.8 35.4 104.9 2.0 102.5 28.2 94.9 16.3 AD-1136229.174.8 21.7 70.0 10.9 58.6 14.0 52.2 1.6 AD-1136230.1 60.7 17.2 77.6 6.454.3 5.5 48.5 8.1 AD-1136231.1 60.2 15.3 64.9 17.5 62.8 25.9 39.2 8.6AD-1136232.1 53.8 19.5 49.8 7.0 71.1 15.0 64.2 12.0 AD-1136233.1 67.810.5 75.0 16.4 99.8 9.7 89.5 18.3 AD-1136234.1 135.9 39.0 99.5 7.3 76.86.8 86.4 32.5 AD-1136235.1 106.5 18.7 97.4 17.9 92.2 11.3 81.5 18.2AD-1136236.1 73.8 11.1 83.1 7.2 85.4 12.0 81.7 5.4 AD-1136237.1 72.045.6 79.6 5.5 58.9 17.0 52.5 7.6 AD-1136238.1 73.1 3.2 75.4 12.1 52.14.1 45.3 7.1 AD-1136239.1 59.5 17.2 66.6 18.3 57.2 8.8 58.9 27.6AD-1136240.1 61.1 7.6 63.8 2.7 89.7 21.7 75.9 6.2 AD-1136241.1 93.9 37.481.2 7.6 74.3 19.1 131.9 66.6 AD-1136242.1 119.9 28.2 142.2 79.5 94.517.8 89.0 65.8 AD-1136243.1 81.2 2.4 69.7 15.0 94.7 32.0 109.6 46.7AD-1136244.1 66.9 32.5 66.8 3.3 67.7 11.6 53.6 1.6 AD-1136245.1 89.625.0 85.4 14.5 79.9 32.3 54.9 11.8 AD-1136246.1 70.6 9.3 67.8 7.7 69.218.8 47.9 14.6 AD-1136247.1 99.4 30.6 69.9 11.6 51.5 14.4 64.9 23.9AD-1136248.1 68.2 10.9 66.0 7.4 82.0 32.8 62.3 9.7 AD-1136249.1 137.153.3 111.3 46.1 66.0 44.0 101.4 105.9 AD-1136250.1 112.9 11.1 143.8 8.3121.6 19.0 104.3 22.7 AD-1136251.1 108.3 30.3 149.1 26.0 135.9 15.9160.4 106.8 AD-1136252.1 94.4 5.2 127.0 37.2 125.2 5.1 97.7 35.3AD-1136253.1 112.3 16.8 130.1 14.8 127.2 23.0 182.7 108.0 AD-1136254.1148.8 15.9 129.8 30.2 140.8 26.9 82.2 50.6 AD-1136255.1 103.0 20.9 119.432.1 141.8 42.9 90.3 54.0 AD-1136256.1 124.3 41.8 108.0 22.9 89.3 27.9118.4 27.6 AD-1136257.1 123.8 50.0 97.2 20.6 104.0 6.6 52.4 7.6AD-1136258.1 61.2 10.2 124.6 23.8 118.5 24.8 117.7 16.9 AD-1136259.189.2 26.4 120.0 20.2 143.7 13.9 98.8 5.8 AD-1136260.1 93.3 22.2 135.422.6 152.5 34.4 150.5 27.6 AD-1136261.1 80.6 17.3 111.9 13.8 125.0 33.0127.6 29.7 AD-1136262.1 71.8 12.8 113.2 7.6 133.9 2.7 104.6 9.0AD-1136263.1 81.0 20.7 93.6 19.1 105.3 24.0 101.2 8.5 AD-1136264.1 73.313.3 82.8 20.6 85.6 8.3 N/A N/A AD-1136265.1 102.0 12.2 122.2 19.5 103.111.1 N/A N/A AD-1136266.1 95.9 22.1 117.0 27.4 120.9 15.0 100.2 22.0AD-1136267.1 102.3 18.1 149.1 13.2 156.4 45.6 197.8 80.6 AD-1136268.195.8 20.1 161.8 23.0 96.2 20.3 N/A N/A AD-1136269.1 83.5 12.5 129.0 9.1113.2 17.3 153.8 51.2 AD-1136270.1 116.6 19.9 112.3 10.5 115.9 21.3 91.927.5 AD-1136271.1 82.9 24.6 81.1 12.3 68.2 20.5 67.0 28.6 AD-1136272.171.3 14.3 86.2 8.9 94.2 15.6 78.1 10.3 AD-1136273.1 77.6 16.4 125.1 10.3129.3 21.4 107.4 17.7 AD-1136274.1 96.3 30.6 169.2 39.0 104.4 19.2 333.6177.6 AD-1136275.1 103.6 13.0 146.3 17.2 97.7 21.7 N/A N/A AD-1136276.1101.7 13.3 143.9 30.2 133.7 27.5 129.2 42.3 AD-1136277.1 82.3 22.6 103.56.1 106.7 33.3 80.3 17.4 AD-1136278.1 69.5 29.9 93.6 24.6 85.4 23.3 72.622.4 AD-1136279.1 64.1 9.0 94.7 12.9 78.5 33.4 76.7 31.1 AD-1136280.174.3 22.3 107.2 15.8 108.0 23.7 85.2 28.9 AD-1136281.1 123.8 23.0 148.230.4 117.0 11.9 135.4 32.4 AD-1136282.1 86.4 24.8 147.3 39.3 153.6 19.2257.4 178.0 AD-1136283.1 63.2 20.2 85.5 26.9 110.6 32.6 N/A N/AAD-1136284.1 84.0 14.1 114.3 11.9 77.9 18.0 208.9 84.2 AD-1136285.1 88.816.1 93.0 29.4 103.1 17.1 82.9 20.6 AD-1136286.1 66.0 10.5 59.8 8.7 51.230.0 60.3 14.0 AD-1136287.1 76.6 25.4 97.0 12.8 89.7 14.3 145.0 110.3AD-1136288.1 73.0 23.3 99.8 28.2 107.0 10.2 86.6 11.2 AD-1136289.1 79.09.3 104.0 20.5 144.6 8.6 141.6 41.6 AD-1136290.1 75.1 17.2 117.4 20.480.4 14.3 N/A N/A AD-1136291.1 50.6 10.3 73.9 19.4 80.5 34.1 N/A N/AAD-1136292.1 65.8 10.9 93.5 14.3 84.7 26.4 113.4 37.8 AD-1136293.1 78.37.4 82.1 9.9 65.6 20.3 71.4 17.4 AD-1136294.1 59.4 7.5 81.6 12.2 62.338.5 114.0 82.5 AD-1136295.1 68.9 9.1 98.9 19.2 115.6 12.7 93.5 16.7AD-1136296.1 85.6 23.0 121.5 13.0 91.4 14.3 107.7 15.6 AD-1136297.1 92.224.2 126.8 24.1 73.8 13.7 218.7 44.7 AD-1136298.1 89.7 11.3 91.1 3.275.8 11.0 N/A N/A AD-1136299.1 75.7 4.3 112.6 26.8 76.8 9.4 98.1 10.5AD-1136300.1 79.9 23.9 99.6 13.2 85.8 28.4 77.2 1.8 AD-1136301.1 81.316.5 58.9 10.8 75.2 18.8 52.9 10.3 AD-1136302.1 69.9 22.1 102.0 40.870.5 23.3 73.7 9.2 AD-1136303.1 73.6 20.6 92.2 16.0 104.6 26.4 103.9 6.0AD-1136304.1 80.4 13.2 108.2 16.5 102.0 12.2 106.9 40.0 AD-1136305.179.2 12.9 114.0 17.1 105.6 28.9 N/A N/A AD-1136306.1 78.0 6.6 111.2 15.3100.5 23.9 N/A N/A AD-1136307.1 76.1 12.7 115.6 28.5 83.8 15.3 96.8 21.5AD-1136308.1 67.4 17.1 66.7 21.3 63.3 17.1 67.4 5.6 AD-1136309.1 65.126.6 79.8 11.5 74.5 4.1 60.7 23.8 AD-1136310.1 71.3 22.6 76.1 16.3 108.935.2 71.2 25.5 AD-1136311.1 59.3 9.8 94.5 8.0 96.2 14.4 89.3 17.7AD-1136312.1 63.7 6.0 105.0 12.9 90.5 5.7 89.3 11.6 AD-1136313.1 75.73.9 105.1 37.5 89.8 21.6 88.8 19.0 AD-1136314.1 108.8 17.9 95.8 12.083.8 21.1 87.0 23.5 AD-1136315.1 92.0 15.8 83.7 15.5 77.1 13.5 73.5 18.1AD-1136316.1 85.1 16.3 66.7 10.9 60.0 8.0 49.3 11.8 AD-1136317.1 64.57.9 72.5 16.7 89.9 21.4 77.8 9.4 AD-1136318.1 88.2 12.5 86.4 13.6 91.017.1 105.4 24.8 AD-1136319.1 63.9 18.5 120.7 38.2 119.4 20.1 92.4 19.3AD-1136320.1 68.9 15.0 100.9 9.9 105.8 27.7 89.6 9.0 AD-1136321.1 96.124.6 91.0 32.8 90.1 16.5 73.3 9.0 AD-1136322.1 101.9 24.1 80.8 18.8 63.511.5 71.3 20.1 AD-1136323.1 73.2 15.8 62.6 22.8 61.0 18.6 68.1 27.7AD-1136324.1 110.1 12.5 88.6 18.2 87.0 9.3 63.6 11.8 AD-1136325.1 83.416.1 90.3 7.4 81.7 17.5 65.5 25.1 AD-1136326.1 87.7 28.8 101.2 12.0 70.315.4 59.2 11.4 AD-1136327.1 142.0 57.3 137.3 10.5 63.4 8.7 85.7 13.0AD-1136328.1 90.4 10.6 85.2 11.8 91.7 31.9 63.8 21.7 AD-1136329.1 79.217.1 89.8 26.0 49.4 9.6 52.0 7.9 AD-1136330.1 125.2 36.0 78.1 21.5 76.59.9 67.1 22.8 AD-1136331.1 100.0 34.1 81.0 2.9 86.7 13.1 64.3 25.9AD-1136332.1 106.9 22.8 72.0 38.2 69.4 23.1 54.3 20.7 AD-1136333.1 76.429.8 110.9 45.1 61.2 8.1 59.3 14.5 AD-1136334.1 105.3 58.3 93.3 14.649.5 19.3 47.3 21.0 AD-1136335.1 71.5 11.5 113.4 24.5 76.3 28.7 70.040.8 AD-1136336.1 136.1 69.8 104.1 23.7 81.2 23.9 57.7 18.1 AD-1136337.187.1 35.7 100.8 20.0 59.4 28.2 38.8 8.9 AD-1136338.1 124.5 31.0 86.814.0 51.3 4.8 77.9 75.1

Example 3. Additional Duplexes Targeting XDH

Additional duplexes targeting the xanthine dehydrogenase (XDH) gene,(human: NCBI refseqID NM_000379; NCBI GeneID: 7498) were designed usingcustom R and Python scripts. The human NM_000379 REFSEQ mRNA, version 4,has a length of 5715 bases.

siRNAs were synthesized and annealed using routine methods known in theart and described above.

Detailed lists of the unmodified XDH sense and antisense strandnucleotide sequences are shown in Table 6. Detailed lists of themodified XDH sense and antisense strand nucleotide sequences are shownin Table 7.

The additional duplexes for screened for activity in vitro.

Free uptake experiments were performed by adding 2.5 W. of siRNAduplexes in PBS per well into a 96 well plate. Complete growth media(47.5 μl) containing about 1.5×10⁴ PMH or PHH was then added to thesiRNA. Cells were incubated for 48 hours prior to RNA purification andRT-qPCR. Single dose experiments in PHH were performed at 500 nM, 100nM, 10 nM and/or 0.1 nM in PHH and in PMH at 500 nM, 100 nM, nM, and 1 Mfinal duplex concentration.

For transfection, PHH cells were grown to near confluence at 37° C. inan atmosphere of 5% CO₂ in Eagle's Minimum Essential Medium (Gibco)supplemented with 10% FBS (ATCC) before being released from the plate bytrypsinization. Transfection was carried out by adding 7.5 μl ofOpti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) to 2.5 μl of each siRNA duplex to anindividual well in a 384-well plate. The mixture was then incubated atroom temperature for 15 minutes. Forty μl of complete growth mediawithout antibiotic containing ˜1.5×10⁴ PHH cells were then added to thesiRNA mixture. Cells were incubated for 24 hours prior to RNApurification. Single dose experiments were performed at 100 nM, 20 nM, 4nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, 0.00128 nM and 0.0001256 nMfinal duplex concentration.

Total RNA isolation was performed using DYNABEADS. Briefly, cells werelysed in 101 of Lysis/Binding Buffer containing 3 μL of beads per welland mixed for 10 minutes on an electrostatic shaker. The washing stepswere automated on a Biotek EL406, using a magnetic plate support. Beadswere washed (in 3 μL) once in Buffer A, once in Buffer B, and twice inBuffer E, with aspiration steps in between. Following a finalaspiration, complete 12 μL RT mixture was added to each well, asdescribed below.

For cDNA synthesis, a master mix of 1.5 μl 10× Buffer, 0.6 μl 10× dNTPs,1.51 Random primers, 0.751 Reverse Transcriptase, 0.751 RNase inhibitorand 9.91 of H₂O per reaction were added per well. Plates were sealed,agitated for 10 minutes on an electrostatic shaker, and then incubatedat 37 degrees C. for 2 hours. Following this, the plates were agitatedat 80 degrees C. for 8 minutes.

RT-qPCR was performed as described above and relative fold change wascalculated as described above.

The results of the free uptake experiments of the dsRNA agents listed inTables 6 and 7 in PHH are shown in Tables 8 and 9. The results of thetransfection experiments of the dsRNA agents listed in Tables 6 and 7 inPHH are shown in Tables 10-12. The IC₅₀ values for the dsRNA agents fromTables 10-12 are included in Table 13.

TABLE 6 Unmodified Sense and Antisense StrandSequences of Xanthine Dehydrogenase dsRNA Agents SEQ SEQ Sense SequenceID Range in Antisense Sequence ID Range in Duplex Name 5′ to 3′ NO:NM_000379.4 5′ to 3′ NO: NM_000379.4 AD-1135990.2 GCACAGUGAUGCUCUCCAAGU  34 228-248 ACUUGGAGAGCAUCACUGUGCAA  393 226-248 AD-1135991.2CACAGUGAUGCUCUCCAAGUU   35 229-249 AACUTGGAGAGCAUCACUGUGCA  394 227-249AD-1297597.1 ACAGUGAUGCUCUCCAAGUAU 1818 230-250 AUACTUGGAGAGCAUCACUGUGC1934 228-250 AD-1297598.1 CAGUGAUGCUCUCCAAGUAUU 1819 231-251AAUACUUGGAGAGCAUCACUGUG 1935 229-251 AD-1297599.1 AGUGAUGCUCUCCAAGUAUGU1820 232-252 ACAUACUUGGAGAGCAUCACUGU 1936 230-252 AD-1297600.1GUGAUGCUCUCCAAGUAUGAU 1821 233-253 AUCAUACUUGGAGAGCAUCACUG 1937 231-253AD-1297601.1 UGAUGCUCUCCAAGUAUGAUU 1822 234-254 AAUCAUACUUGGAGAGCAUCACU1938 232-254 AD-1135992.2 GAUGCUCUCCAAGUAUGAUCU   36 235-255AGAUCAUACUUGGAGAGCAUCAC  395 233-255 AD-1135993.2 AUGCUCUCCAAGUAUGAUCGU  37 236-256 ACGAUCAUACUUGGAGAGCAUCA  396 234-256 AD-1135994.2UGCUCUCCAAGUAUGAUCGUU   38 237-257 AACGAUCAUACUUGGAGAGCAUC  397 235-257AD-1135995.2 GCUCUCCAAGUAUGAUCGUCU   39 238-258 AGACGAUCAUACUUGGAGAGCAU 398 236-258 AD-1135996.2 CUCUCCAAGUAUGAUCGUCUU   40 239-259AAGACGAUCAUACUUGGAGAGCA  399 237-259 AD-1297602.1 UCUCCAAGUAUGAUCGUCUGU1823 240-260 ACAGACGAUCAUACUUGGAGAGC 1939 238-260 AD-1297603.1CUCCAAGUAUGAUCGUCUGCU 1824 241-261 AGCAGACGAUCAUACUUGGAGAG 1940 239-261AD-1297604.1 UCCAAGUAUGAUCGUCUGCAU 1825 242-262 AUGCAGACGAUCAUACUUGGAGA1941 240-262 AD-1297605.1 CCAAGUAUGAUCGUCUGCAGU 1826 243-263ACUGCAGACGAUCAUACUUGGAG 1942 241-263 AD-1297606.1 CAAGUAUGAUCGUCUGCAGAU1827 244-264 AUCUGCAGACGAUCAUACUUGGA 1943 242-264 AD-1297607.1AAGUAUGAUCGUCUGCAGAAU 1828 245-265 AUUCTGCAGACGAUCAUACUUGG 1944 243-265AD-1297608.1 AGUAUGAUCGUCUGCAGAACU 1829 246-266 AGUUCUGCAGACGAUCAUACUUG1945 244-266 AD-1297609.1 GUAUGAUCGUCUGCAGAACAU 1830 247-267AUGUTCTGCAGACGAUCAUACUU 1946 245-267 AD-1297610.1 UAUGAUCGUCUGCAGAACAAU1831 248-268 AUUGTUCUGCAGACGAUCAUACU 1947 246-268 AD-1297611.1AUGAUCGUCUGCAGAACAAGU 1832 249-269 ACUUGUUCUGCAGACGAUCAUAC 1948 247-269AD-1136037.2 GGGAGUAUUUCUCAGCAUUCU   81 1320-1340AGAATGCUGAGAAAUACUCCCCC  440 1318-1340 AD-1136038.2GGAGUAUUUCUCAGCAUUCAU   82 1321-1341 AUGAAUGCUGAGAAAUACUCCCC  4411319-1341 AD-1136039.2 GAGUAUUUCUCAGCAUUCAAU   83 1322-1342AUUGAAUGCUGAGAAAUACUCCC  442 1320-1342 AD-1136040.2AGUAUUUCUCAGCAUUCAAGU   84 1323-1343 ACUUGAAUGCUGAGAAAUACUCC  4431321-1343 AD-1136041.2 GUAUUUCUCAGCAUUCAAGCU   85 1324-1344AGCUTGAAUGCUGAGAAAUACUC  444 1322-1344 AD-1297612.1UAUUUCUCAGCAUUCAAGCAU 1833 1325-1345 AUGCTUGAAUGCUGAGAAAUACU 19491323-1345 AD-1297613.1 AUUUCUCAGCAUUCAAGCAGU 1834 1326-1346ACUGCUUGAAUGCUGAGAAAUAC 1950 1324-1346 AD-1297614.1UUUCUCAGCAUUCAAGCAGGU 1835 1327-1347 ACCUGCTUGAAUGCUGAGAAAUA 19511325-1347 AD-1297615.1 UUCUCAGCAUUCAAGCAGGCU 1836 1328-1348AGCCTGCUUGAAUGCUGAGAAAU 1952 1326-1348 AD-1297616.1UCUCAGCAUUCAAGCAGGCCU 1837 1329-1349 AGGCCUGCUUGAAUGCUGAGAAA 19531327-1349 AD-1297617.1 CUCAGCAUUCAAGCAGGCCUU 1838 1330-1350AAGGCCTGCUUGAAUGCUGAGAA 1954 1328-1350 AD-1297618.1UCAGCAUUCAAGCAGGCCUCU 1839 1331-1351 AGAGGCCUGCUUGAAUGCUGAGA 19551329-1351 AD-1297619.1 CAGCAUUCAAGCAGGCCUCCU 1840 1332-1352AGGAGGCCUGCUUGAAUGCUGAG 1956 1330-1352 AD-1297620.1AAGAAGGUUCCAGGGUUUGUU 1841 1955-1975 AACAAACCCUGGAACCUUCUUAG 19571953-1975 AD-1297621.1 AGAAGGUUCCAGGGUUUGUUU 1842 1956-1976AAACAAACCCUGGAACCUUCUUA 1958 1954-1976 AD-1297622.1GAAGGUUCCAGGGUUUGUUUU 1843 1957-1977 AAAACAAACCCUGGAACCUUCUU 19591955-1977 AD-1297623.1 AAGGUUCCAGGGUUUGUUUGU 1844 1958-1978ACAAACAAACCCUGGAACCUUCU 1960 1956-1978 AD-1297624.1AGGUUCCAGGGUUUGUUUGUU 1845 1959-1979 AACAAACAAACCCUGGAACCUUC 19611957-1979 AD-1297625.1 GGUUCCAGGGUUUGUUUGUUU 1846 1960-1980AAACAAACAAACCCUGGAACCUU 1962 1958-1980 AD-1297626.1GUUCCAGGGUUUGUUUGUUUU 1847 1961-1981 AAAACAAACAAACCCUGGAACCU 19631959-1981 AD-1136050.2 UUCCAGGGUUUGUUUGUUUCU   94 1962-1982AGAAACAAACAAACCCUGGAACC  453 1960-1982 AD-1297627.1UCCAGGGUUUGUUUGUUUCAU 1848 1963-1983 AUGAAACAAACAAACCCUGGAAC 19641961-1983 AD-1297628.1 CCAGGGUUUGUUUGUUUCAUU 1849 1964-1984AAUGAAACAAACAAACCCUGGAA 1965 1962-1984 AD-1136051.2CAGGGUUUGUUUGUUUCAUUU   95 1965-1985 AAAUGAAACAAACAAACCCUGGA  4541963-1985 AD-1136052.2 GGGUUUGUUUGUUUCAUUUCU   96 1967-1987AGAAAUGAAACAAACAAACCCUG  455 1965-1987 AD-1136053.2GGUUUGUUUGUUUCAUUUCCU   97 1968-1988 AGGAAAUGAAACAAACAAACCCU  4561966-1988 AD-1297629.1 GUUUGUUUGUUUCAUUUCCGU 1850 1969-1989ACGGAAAUGAAACAAACAAACCC 1966 1967-1989 AD-1297630.1UUUGUUUGUUUCAUUUCCGCU 1851 1970-1990 AGCGGAAAUGAAACAAACAAACC 19671968-1990 AD-1136054.2 UUGUUUGUUUCAUUUCCGCUU   98 1971-1991AAGCGGAAAUGAAACAAACAAAC  457 1969-1991 AD-1297631.1UGUUUGUUUCAUUUCCGCUGU 1852 1972-1992 ACAGCGGAAAUGAAACAAACAAA 19681970-1992 AD-1297632.1 GUUUGUUUCAUUUCCGCUGAU 1853 1973-1993AUCAGCGGAAAUGAAACAAACAA 1969 1971-1993 AD-1297633.1UUUGUUUCAUUUCCGCUGAUU 1854 1974-1994 AAUCAGCGGAAAUGAAACAAACA 19701972-1994 AD-1297634.1 UUGUUUCAUUUCCGCUGAUGU 1855 1975-1995ACAUCAGCGGAAAUGAAACAAAC 1971 1973-1995 AD-1297635.1UGUUUCAUUUCCGCUGAUGAU 1856 1976-1996 AUCATCAGCGGAAAUGAAACAAA 19721974-1996 AD-1297636.1 GUUUCAUUUCCGCUGAUGAUU 1857 1977-1997AAUCAUCAGCGGAAAUGAAACAA 1973 1975-1997 AD-1297637.1UUUCAUUUCCGCUGAUGAUGU 1858 1978-1998 ACAUCAUCAGCGGAAAUGAAACA 19741976-1998 AD-1297638.1 GGAGAUGGAGCUCUUUGUGUU 1859 2353-2373AACACAAAGAGCUCCAUCUCCCC 1975 2351-2373 AD-1297639.1GAGAUGGAGCUCUUUGUGUCU 1860 2354-2374 AGACACAAAGAGCUCCAUCUCCC 19762352-2374 AD-1297640.1 AGAUGGAGCUCUUUGUGUCUU 1861 2355-2375AAGACACAAAGAGCUCCAUCUCC 1977 2353-2375 AD-1297641.1GAUGGAGCUCUUUGUGUCUAU 1862 2356-2376 AUAGACACAAAGAGCUCCAUCUC 19782354-2376 AD-1297642.1 AUGGAGCUCUUUGUGUCUACU 1863 2357-2377AGUAGACACAAAGAGCUCCAUCU 1979 2355-2377 AD-1297643.1UGGAGCUCUUUGUGUCUACAU 1864 2358-2378 AUGUAGACACAAAGAGCUCCAUC 19802356-2378 AD-1297644.1 GGAGCUCUUUGUGUCUACACU 1865 2359-2379AGUGTAGACACAAAGAGCUCCAU 1981 2357-2379 AD-1297645.1GAGCUCUUUGUGUCUACACAU 1866 2360-2380 AUGUGUAGACACAAAGAGCUCCA 19822358-2380 AD-1136073.2 AGCUCUUUGUGUCUACACAGU  117 2361-2381ACUGTGTAGACACAAAGAGCUCC  476 2359-2381 AD-1136074.2GCUCUUUGUGUCUACACAGAU  118 2362-2382 AUCUGUGUAGACACAAAGAGCUC  4772360-2382 AD-1136075.2 CUCUUUGUGUCUACACAGAAU  119 2363-2383AUUCTGTGUAGACACAAAGAGCU  478 2361-2383 AD-1136076.2UCUUUGUGUCUACACAGAACU  120 2364-2384 AGUUCUGUGUAGACACAAAGAGC  4792362-2384 AD-1136077.2 CUUUGUGUCUACACAGAACAU  121 2365-2385AUGUTCTGUGUAGACACAAAGAG  480 2363-2385 AD-1136078.2UUUGUGUCUACACAGAACACU  122 2366-2386 AGUGTUCUGUGUAGACACAAAGA  4812364-2386 AD-1297646.1 UUGUGUCUACACAGAACACCU 1867 2367-2387AGGUGUTCUGUGUAGACACAAAG 1983 2365-2387 AD-1297647.1UGUGUCUACACAGAACACCAU 1868 2368-2388 AUGGTGTUCUGUGUAGACACAAA 19842366-2388 AD-1297648.1 GUGUCUACACAGAACACCAUU 1869 2369-2389AAUGGUGUUCUGUGUAGACACAA 1985 2367-2389 AD-1297649.1UGUCUACACAGAACACCAUGU 1870 2370-2390 ACAUGGTGUUCUGUGUAGACACA 19862368-2390 AD-1297650.1 GUCUACACAGAACACCAUGAU 1871 2371-2391AUCATGGUGUUCUGUGUAGACAC 1987 2369-2391 AD-1297651.1UCUACACAGAACACCAUGAAU 1872 2372-2392 AUUCAUGGUGUUCUGUGUAGACA 19882370-2392 AD-1297652.1 CUACACAGAACACCAUGAAGU 1873 2373-2393ACUUCAUGGUGUUCUGUGUAGAC 1989 2371-2393 AD-1297653.1UACACAGAACACCAUGAAGAU 1874 2374-2394 AUCUTCAUGGUGUUCUGUGUAGA 19902372-2394 AD-1297654.1 GGGAACACCCAGGAUCUCUCU 1875 2681-2701AGAGAGAUCCUGGGUGUUCCCCA 1991 2679-2701 AD-1297655.1GGAACACCCAGGAUCUCUCUU 1876 2682-2702 AAGAGAGAUCCUGGGUGUUCCCC 19922680-2702 AD-1297656.1 GAACACCCAGGAUCUCUCUCU 1877 2683-2703AGAGAGAGAUCCUGGGUGUUCCC 1993 2681-2703 AD-1297657.1AACACCCAGGAUCUCUCUCAU 1878 2684-2704 AUGAGAGAGAUCCUGGGUGUUCC 19942682-2704 AD-1297658.1 ACACCCAGGAUCUCUCUCAGU 1879 2685-2705ACUGAGAGAGAUCCUGGGUGUUC 1995 2683-2705 AD-1297659.1CACCCAGGAUCUCUCUCAGAU 1880 2686-2706 AUCUGAGAGAGAUCCUGGGUGUU 19962684-2706 AD-1297660.1 ACCCAGGAUCUCUCUCAGAGU 1881 2687-2707ACUCTGAGAGAGAUCCUGGGUGU 1997 2685-2707 AD-1297661.1CCCAGGAUCUCUCUCAGAGUU 1882 2688-2708 AACUCUGAGAGAGAUCCUGGGUG 19982686-2708 AD-1297662.1 CCAGGAUCUCUCUCAGAGUAU 1883 2689-2709AUACTCTGAGAGAGAUCCUGGGU 1999 2687-2709 AD-1297663.1CAGGAUCUCUCUCAGAGUAUU 1884 2690-2710 AAUACUCUGAGAGAGAUCCUGGG 20002688-2710 AD-1136082.2 AGGAUCUCUCUCAGAGUAUUU  126 2691-2711AAAUACTCUGAGAGAGAUCCUGG  485 2689-2711 AD-1136083.2GGAUCUCUCUCAGAGUAUUAU  127 2692-2712 AUAATACUCUGAGAGAGAUCCUG  4862690-2712 AD-1136084.2 GAUCUCUCUCAGAGUAUUAUU  128 2693-2713AAUAAUACUCUGAGAGAGAUCCU  487 2691-2713 AD-1136085.2AUCUCUCUCAGAGUAUUAUGU  129 2694-2714 ACAUAAUACUCUGAGAGAGAUCC  4882692-2714 AD-1136086.2 UCUCUCUCAGAGUAUUAUGGU  130 2695-2715ACCAUAAUACUCUGAGAGAGAUC  489 2693-2715 AD-1136087.2CUCUCUCAGAGUAUUAUGGAU  131 2696-2716 AUCCAUAAUACUCUGAGAGAGAU  4902694-2716 AD-1136088.2 UCUCUCAGAGUAUUAUGGAAU  132 2697-2717AUUCCAUAAUACUCUGAGAGAGA  491 2695-2717 AD-1136089.2CUCUCAGAGUAUUAUGGAACU  133 2698-2718 AGUUCCAUAAUACUCUGAGAGAG  4922696-2718 AD-1136090.2 UCUCAGAGUAUUAUGGAACGU  134 2699-2719ACGUUCCAUAAUACUCUGAGAGA  493 2697-2719 AD-1297664.1CUCAGAGUAUUAUGGAACGAU 1885 2700-2720 AUCGTUCCAUAAUACUCUGAGAG 20012698-2720 AD-1136091.2 UCAGAGUAUUAUGGAACGAGU  135 2701-2721ACUCGUUCCAUAAUACUCUGAGA  494 2699-2721 AD-1136092.2CAGAGUAUUAUGGAACGAGCU  136 2702-2722 AGCUCGUUCCAUAAUACUCUGAG  4952700-2722 AD-1297665.1 AGAGUAUUAUGGAACGAGCUU 1886 2703-2723AAGCTCGUUCCAUAAUACUCUGA 2002 2701-2723 AD-1297666.1GAGUAUUAUGGAACGAGCUUU 1887 2704-2724 AAAGCUCGUUCCAUAAUACUCUG 20032702-2724 AD-1297667.1 AGUAUUAUGGAACGAGCUUUU 1888 2705-2725AAAAGCTCGUUCCAUAAUACUCU 2004 2703-2725 AD-1297668.1GUAUUAUGGAACGAGCUUUAU 1889 2706-2726 AUAAAGCUCGUUCCAUAAUACUC 20052704-2726 AD-1297669.1 UAUUAUGGAACGAGCUUUAUU 1890 2707-2727AAUAAAGCUCGUUCCAUAAUACU 2006 2705-2727 AD-1297670.1AUUAUGGAACGAGCUUUAUUU 1891 2708-2728 AAAUAAAGCUCGUUCCAUAAUAC 20072706-2728 AD-1297671.1 UUAUGGAACGAGCUUUAUUCU 1892 2709-2729AGAAUAAAGCUCGUUCCAUAAUA 2008 2707-2729 AD-1297672.1UAUGGAACGAGCUUUAUUCCU 1893 2710-2730 AGGAAUAAAGCUCGUUCCAUAAU 20092708-2730 AD-1297673.1 CUCUUCCUGGCUGCUUCUAUU 1894 3869-3889AAUAGAAGCAGCCAGGAAGAGGG 2010 3867-3889 AD-1297674.1UCUUCCUGGCUGCUUCUAUCU 1895 3870-3890 AGAUAGAAGCAGCCAGGAAGAGG 20113868-3890 AD-1297675.1 CUUCCUGGCUGCUUCUAUCUU 1896 3871-3891AAGAUAGAAGCAGCCAGGAAGAG 2012 3869-3891 AD-1136159.2UUCCUGGCUGCUUCUAUCUUU  202 3872-3892 AAAGAUAGAAGCAGCCAGGAAGA  5613870-3892 AD-1136160.2 UCCUGGCUGCUUCUAUCUUCU  203 3873-3893AGAAGAUAGAAGCAGCCAGGAAG  562 3871-3893 AD-1136161.2CCUGGCUGCUUCUAUCUUCUU  204 3874-3894 AAGAAGAUAGAAGCAGCCAGGAA  5633872-3894 AD-1136162.2 CUGGCUGCUUCUAUCUUCUUU  205 3875-3895AAAGAAGAUAGAAGCAGCCAGGA  564 3873-3895 AD-1136163.2UGGCUGCUUCUAUCUUCUUUU  206 3876-3896 AAAAGAAGAUAGAAGCAGCCAGG  5653874-3896 AD-1297676.1 GGCUGCUUCUAUCUUCUUUGU 1897 3877-3897ACAAAGAAGAUAGAAGCAGCCAG 2013 3875-3897 AD-1136164.2GCUGCUUCUAUCUUCUUUGCU  207 3878-3898 AGCAAAGAAGAUAGAAGCAGCCA  5663876-3898 AD-1136165.2 CUGCUUCUAUCUUCUUUGCCU  208 3879-3899AGGCAAAGAAGAUAGAAGCAGCC  567 3877-3899 AD-1136166.2UGCUUCUAUCUUCUUUGCCAU  209 3880-3900 AUGGCAAAGAAGAUAGAAGCAGC  5683878-3900 AD-1136167.2 GCUUCUAUCUUCUUUGCCAUU  210 3881-3901AAUGGCAAAGAAGAUAGAAGCAG  569 3879-3901 AD-1136168.2CUUCUAUCUUCUUUGCCAUCU  211 3882-3902 AGAUGGCAAAGAAGAUAGAAGCA  5703880-3902 AD-1136169.2 UUCUAUCUUCUUUGCCAUCAU  212 3883-3903AUGATGGCAAAGAAGAUAGAAGC  571 3881-3903 AD-1136170.2UCUAUCUUCUUUGCCAUCAAU  213 3884-3904 AUUGAUGGCAAAGAAGAUAGAAG  5723882-3904 AD-1136171.2 CUAUCUUCUUUGCCAUCAAAU  214 3885-3905AUUUGATGGCAAAGAAGAUAGAA  573 3883-3905 AD-1136172.2UAUCUUCUUUGCCAUCAAAGU  215 3886-3906 ACUUUGAUGGCAAAGAAGAUAGA  5743884-3906 AD-1136173.2 AUCUUCUUUGCCAUCAAAGAU  216 3887-3907AUCUTUGAUGGCAAAGAAGAUAG  575 3885-3907 AD-1297677.1UCUUCUUUGCCAUCAAAGAUU 1898 3888-3908 AAUCUUUGAUGGCAAAGAAGAUA 20143886-3908 AD-1136174.2 CUUCUUUGCCAUCAAAGAUGU  217 3889-3909ACAUCUUUGAUGGCAAAGAAGAU  576 3887-3909 AD-1297678.1UUCUUUGCCAUCAAAGAUGCU 1899 3890-3910 AGCATCTUUGAUGGCAAAGAAGA 20153888-3910 AD-1297679.1 UCUUUGCCAUCAAAGAUGCCU 1900 3891-3911AGGCAUCUUUGAUGGCAAAGAAG 2016 3889-3911 AD-1297680.1CUUUGCCAUCAAAGAUGCCAU 1901 3892-3912 AUGGCATCUUUGAUGGCAAAGAA 20173890-3912 AD-1297681.1 UUUGCCAUCAAAGAUGCCAUU 1902 3893-3913AAUGGCAUCUUUGAUGGCAAAGA 2018 3891-3913 AD-1297682.1UUGCCAUCAAAGAUGCCAUCU 1903 3894-3914 AGAUGGCAUCUUUGAUGGCAAAG 20193892-3914 AD-1297683.1 UGCCAUCAAAGAUGCCAUCCU 1904 3895-3915AGGATGGCAUCUUUGAUGGCAAA 2020 3893-3915 AD-1297684.1GCCAUCAAAGAUGCCAUCCGU 1905 3896-3916 ACGGAUGGCAUCUUUGAUGGCAA 20213894-3916 AD-1297685.1 AAACCAAUGAACAGCAAAGCU 1906 4512-4532AGCUTUGCUGUUCAUUGGUUUGA 2022 4510-4532 AD-1297686.1AACCAAUGAACAGCAAAGCAU 1907 4513-4533 AUGCTUTGCUGUUCAUUGGUUUG 20234511-4533 AD-1297687.1 ACCAAUGAACAGCAAAGCAUU 1908 4514-4534AAUGCUTUGCUGUUCAUUGGUUU 2024 4512-4534 AD-1297688.1CCAAUGAACAGCAAAGCAUAU 1909 4515-4535 AUAUGCTUUGCUGUUCAUUGGUU 20254513-4535 AD-1297689.1 CAAUGAACAGCAAAGCAUAAU 1910 4516-4536AUUATGCUUUGCUGUUCAUUGGU 2026 4514-4536 AD-1297690.1AAUGAACAGCAAAGCAUAACU 1911 4517-4537 AGUUAUGCUUUGCUGUUCAUUGG 20274515-4537 AD-1297691.1 AUGAACAGCAAAGCAUAACCU 1912 4518-4538AGGUTATGCUUUGCUGUUCAUUG 2028 4516-4538 AD-1297692.1UGAACAGCAAAGCAUAACCUU 1913 4519-4539 AAGGUUAUGCUUUGCUGUUCAUU 20294517-4539 AD-1136221.2 GAACAGCAAAGCAUAACCUUU  264 4520-4540AAAGGUUAUGCUUUGCUGUUCAU  623 4518-4540 AD-1136222.2AACAGCAAAGCAUAACCUUGU  265 4521-4541 ACAAGGTUAUGCUUUGCUGUUCA  6244519-4541 AD-1297693.1 ACAGCAAAGCAUAACCUUGAU 1914 4522-4542AUCAAGGUUAUGCUUUGCUGUUC 2030 4520-4542 AD-1297694.1CAGCAAAGCAUAACCUUGAAU 1915 4523-4543 AUUCAAGGUUAUGCUUUGCUGUU 20314521-4543 AD-1136223.2 AGCAAAGCAUAACCUUGAAUU  266 4524-4544AAUUCAAGGUUAUGCUUUGCUGU  625 4522-4544 AD-1136224.2GCAAAGCAUAACCUUGAAUCU  267 4525-4545 AGAUTCAAGGUUAUGCUUUGCUG  6264523-4545 AD-1136225.2 CAAAGCAUAACCUUGAAUCUU  268 4526-4546AAGATUCAAGGUUAUGCUUUGCU  627 4524-4546 AD-1297695.1AAAGCAUAACCUUGAAUCUAU 1916 4527-4547 AUAGAUTCAAGGUUAUGCUUUGC 20324525-4547 AD-1297696.1 AAGCAUAACCUUGAAUCUAUU 1917 4528-4548AAUAGAUUCAAGGUUAUGCUUUG 2033 4526-4548 AD-1297697.1AGCAUAACCUUGAAUCUAUAU 1918 4529-4549 AUAUAGAUUCAAGGUUAUGCUUU 20344527-4549 AD-1136226.2 GCAUAACCUUGAAUCUAUACU  269 4530-4550AGUAUAGAUUCAAGGUUAUGCUU  628 4528-4550 AD-1136227.2CAUAACCUUGAAUCUAUACUU  270 4531-4551 AAGUAUAGAUUCAAGGUUAUGCU  6294529-4551 AD-1136228.2 AUAACCUUGAAUCUAUACUCU  271 4532-4552AGAGUAUAGAUUCAAGGUUAUGC  630 4530-4552 AD-1136229.2UAACCUUGAAUCUAUACUCAU  272 4533-4553 AUGAGUAUAGAUUCAAGGUUAUG  6314531-4553 AD-1136230.2 AACCUUGAAUCUAUACUCAAU  273 4534-4554AUUGAGTAUAGAUUCAAGGUUAU  632 4532-4554 AD-1136231.2ACCUUGAAUCUAUACUCAAAU  274 4535-4555 AUUUGAGUAUAGAUUCAAGGUUA  6334533-4555 AD-1136232.2 CCUUGAAUCUAUACUCAAAUU  275 4536-4556AAUUTGAGUAUAGAUUCAAGGUU  634 4534-4556 AD-1297698.1CUUGAAUCUAUACUCAAAUUU 1919 4537-4557 AAAUTUGAGUAUAGAUUCAAGGU 20354535-4557 AD-1297699.1 UUGAAUCUAUACUCAAAUUUU 1920 4538-4558AAAAUUUGAGUAUAGAUUCAAGG 2036 4536-4558 AD-1297700.1GAAUCUAUACUCAAAUUUUGU 1921 4540-4560 ACAAAAUUUGAGUAUAGAUUCAA 20374538-4560 AD-1136233.2 AAUCUAUACUCAAAUUUUGCU  276 4541-4561AGCAAAAUUUGAGUAUAGAUUCA  635 4539-4561 AD-1136234.2AUCUAUACUCAAAUUUUGCAU  277 4542-4562 AUGCAAAAUUUGAGUAUAGAUUC  6364540-4562 AD-1297701.1 UCUAUACUCAAAUUUUGCAAU 1922 4543-4563AUUGCAAAAUUUGAGUAUAGAUU 2038 4541-4563 AD-1297702.1CUAUACUCAAAUUUUGCAAUU 1923 4544-4564 AAUUGCAAAAUUUGAGUAUAGAU 20394542-4564 AD-1297703.1 UAUACUCAAAUUUUGCAAUGU 1924 4545-4565ACAUTGCAAAAUUUGAGUAUAGA 2040 4543-4565 AD-1297704.1AUACUCAAAUUUUGCAAUGAU 1925 4546-4566 AUCATUGCAAAAUUUGAGUAUAG 20414544-4566 AD-1297705.1 UACUCAAAUUUUGCAAUGAGU 1926 4547-4567ACUCAUUGCAAAAUUUGAGUAUA 2042 4545-4567 AD-1297706.1ACUCAAAUUUUGCAAUGAGGU 1927 4548-4568 ACCUCAUUGCAAAAUUUGAGUAU 20434546-4568 AD-1297707.1 CUCAAAUUUUGCAAUGAGGCU 1928 4549-4569AGCCTCAUUGCAAAAUUUGAGUA 2044 4547-4569 AD-1297708.1UCAAAUUUUGCAAUGAGGCAU 1929 4550-4570 AUGCCUCAUUGCAAAAUUUGAGU 20454548-4570 AD-1297709.1 CAAAUUUUGCAAUGAGGCAGU 1930 4551-4571ACUGCCTCAUUGCAAAAUUUGAG 2046 4549-4571 AD-1297710.1AAAUUUUGCAAUGAGGCAGUU 1931 4552-4572 AACUGCCUCAUUGCAAAAUUUGA 20474550-4572 AD-1297711.1 AAUUUUGCAAUGAGGCAGUGU 1932 4553-4573ACACTGCCUCAUUGCAAAAUUUG 2048 4551-4573 AD-1297712.1AUUUUGCAAUGAGGCAGUGGU 1933 4554-4574 ACCACUGCCUCAUUGCAAAAUUU 20494552-4574 AD-1395794.1 CUCUCCAAGUAUGAUCGUCUU   40 239-259AAGACGAUCAUACUUGGAGAGCG 2050 237-259 AD-1395797.1 AGGAUCUCUCUCAGAGUAUUU 126 2691-2711 AAAUACTCUGAGAGAGAUCCUGG  485 2689-2711 AD-1395803.1CAGUGAUGCUCUCCAAGUAUU 1819 231-251 AAUACUTGGAGAGCAUCACUGUG 2051 229-251AD-1395805.1 CCAAGUAUGAUCGUCUGCAGU 1826 243-263 ACUGCAGACGATCAUACUUGGAG2052 241-263 AD-1395807.1 GUUUGUUUCAUUUCCGCUGAU 1853 1973-1993ATCAGCGGAAATGAAACAAACGG 2053 1971-1993 AD-1395811.1AACACCCAGGAUCUCUCUCAU 1878 2684-2704 ATGAGAGAGAUCCUGGGUGUUCC 20542682-2704 AD-1395816.1 GGGUUUGUUUGUUUCAUUUCU   96 1967-1987AGAAAUGAAACAAACAAACCCUG  455 1965-1987 AD-1395823.1GAGUAUUUCUCAGCAUUCAAU   83 1322-1342 ATUGAATGCUGAGAAAUACUCCC 20551320-1342

TABLE 7 Modified Sense and Antisense StrandSequences of Xanthine Dehydrogenase dsRNA Agents SEQ SEQ SEQ DuplexSense Sequence ID Antisense Sequence ID mRNA Target ID Name 5′ to 3′ NO:5′ to 3′ NO: Sequence NO: AD- gscsacagUfgAfUfGfcucuccaaguL96  752asCfsuudGg(Agn)gag 1111 UUGCACAGUGAUGCUCUCCAAG 1470 1135990.2cauCfaCfugugcsasa U AD- csascaguGfaUfGfCfucuccaaguuL96  753asAfscudTg(G2p)aga 1112 UGCACAGUGAUGCUCUCCAAGU 1471 1135991.2gcaUfcAfcugugscsa A AD- ascsagugAfuGfCfUfcuccaaguauL96 2056asUfsacdTu(G2p)gag 2180 GCACAGUGAUGCUCUCCAAGUA 2304 1297597.1agcAfuCfacugusgsc U AD- csasgugaUfgCfUfCfuccaaguauuL96 2057asAfsuacUfuggagagC 2181 CACAGUGAUGCUCUCCAAGUAU 2305 1297598.1faUfcacugsusg G AD- asgsugauGfcUfCfUfccaaguauguL96 2058asCfsauaCfuuggagaG 2182 ACAGUGAUGCUCUCCAAGUAUG 2306 1297599.1fcAfucacusgsu A AD- gsusgaugCfuCfUfCfcaaguaugauL96 2059asUfscauAfcuuggagA 2183 CAGUGAUGCUCUCCAAGUAUGA 2307 1297600.1fgCfaucacsusg U AD- usgsaugcUfcUfCfCfaaguaugauuL96 2060asAfsucaUfacuuggaG 2184 AGUGAUGCUCUCCAAGUAUGAU 2308 1297601.1faGfcaucascsu C AD- gsasugcuCfuCfCfAfaguaugaucuL96  754asGfsaucAfuacuuggA 1113 GUGAUGCUCUCCAAGUAUGAUC 1472 1135992.2fgAfgcaucsasc G AD- asusgcucUfcCfAfAfguaugaucguL96  755asCfsgauCfauacuugG 1114 UGAUGCUCUCCAAGUAUGAUCG 1473 1135993.2faGfagcauscsa U AD- usgscucuCfcAfAfGfuaugaucguuL96  756asAfscgaUfcauacuuG 1115 GAUGCUCUCCAAGUAUGAUCGU 1474 1135994.2fgAfgagcasusc C AD- gscsucucCfaAfGfUfaugaucgucuL96  757asGfsacgAfucauacuU 1116 AUGCUCUCCAAGUAUGAUCGUC 1475 1135995.2fgGfagagcsasu U AD- csuscuccAfaGfUfAfugaucgucuuL96  758asAfsgacGfaucauacU 1117 UGCUCUCCAAGUAUGAUCGUCU 1476 1135996.2fuGfgagagscsa G AD- uscsuccaAfgUfAfUfgaucgucuguL96 2061asCfsagaCfgaucauaC 2185 GCUCUCCAAGUAUGAUCGUCUG 2309 1297602.1fuUfggagasgsc C AD- csusccaaGfuAfUfGfaucgucugcuL96 2062asGfscadGa(C2p)gau 2186 CUCUCCAAGUAUGAUCGUCUGC 2310 1297603.1cauAfcUfuggagsasg A AD- uscscaagUfaUfGfAfucgucugcauL96 2063asUfsgcdAg(Agn)cga 2187 UCUCCAAGUAUGAUCGUCUGCA 2311 1297604.1ucaUfaCfuuggasgsa G AD- cscsaaguAfuGfAfUfcgucugcaguL96 2064asCfsugcAfgacgaucA 2188 CUCCAAGUAUGAUCGUCUGCAG 2312 1297605.1fuAfcuuggsasg A AD- csasaguaUfgAfUfCfgucugcagauL96 2065asUfscudGc(Agn)gac 2189 UCCAAGUAUGAUCGUCUGCAGA 2313 1297606.1gauCfaUfacuugsgsa A AD- asasguauGfaUfCfGfucugcagaauL96 2066asUfsucdTg(C2p)aga 2190 CCAAGUAUGAUCGUCUGCAGAA 2314 1297607.1cgaUfcAfuacuusgsg C AD- asgsuaugAfuCfGfUfcugcagaacuL96 2067asGfsuudCu(G2p)cag 2191 CAAGUAUGAUCGUCUGCAGAAC 2315 1297608.1acgAfuCfauacususg A AD- gsusaugaUfcGfUfCfugcagaacauL96 2068asUfsgudTc(Tgn)gca 2192 AAGUAUGAUCGUCUGCAGAACA 2316 1297609.1gacGfaUfcauacsusu A AD- usasugauCfgUfCfUfgcagaacaauL96 2069asUfsugdTu(C2p)ugc 2193 AGUAUGAUCGUCUGCAGAACAA 2317 1297610.1agaCfgAfucauascsu G AD- asusgaucGfuCfUfGfcagaacaaguL96 2070asCfsuugUfucugcagA 2194 GUAUGAUCGUCUGCAGAACAAG 2318 1297611.1fcGfaucausasc A AD- gsgsgaguAfuUfUfCfucagcauucuL96  799asGfsaadTg(C2p)uga 1158 GGGGGAGUAUUUCUCAGCAUUC 1517 1136037.2gaaAfuAfcucccscsc A AD- gsgsaguaUfuUfCfUfcagcauucauL96  800asUfsgadAu(G2p)cug 1159 GGGGAGUAUUUCUCAGCAUUCA 1518 1136038.2agaAfaUfacuccscsc A AD- gsasguauUfuCfUfCfagcauucaauL96  801asUfsugaAfugcugagA 1160 GGGAGUAUUUCUCAGCAUUCAA 1519 1136039.2faAfuacucscsc G AD- asgsuauuUfcUfCfAfgcauucaaguL96  802asCfsuugAfaugcugaG 1161 GGAGUAUUUCUCAGCAUUCAAG 1520 1136040.2faAfauacuscsc C AD- gsusauuuCfuCfAfGfcauucaagcuL96  803asGfscudTg(Agn)aug 1162 GAGUAUUUCUCAGCAUUCAAGC 1521 1136041.2cugAfgAfaauacsusc A AD- usasuuucUfcAfGfCfauucaagcauL96 2071asUfsgcdTu(G2p)aau 2195 AGUAUUUCUCAGCAUUCAAGCA 2319 1297612.1gcuGfaGfaaauascsu G AD- asusuucuCfaGfCfAfuucaagcaguL96 2072asCfsugcUfugaaugcU 2196 GUAUUUCUCAGCAUUCAAGCAG 2320 1297613.1fgAfgaaausasc G AD- ususucucAfgCfAfUfucaagcagguL96 2073asCfscudGc(Tgn)uga 2197 UAUUUCUCAGCAUUCAAGCAGG 2321 1297614.1augCfuGfagaaasusa C AD- ususcucaGfcAfUfUfcaagcaggcuL96 2074asGfsccdTg(C2p)uug 2198 AUUUCUCAGCAUUCAAGCAGGC 2322 1297615.1aauGfcUfgagaasasu C AD- uscsucagCfaUfUfCfaagcaggccuL96 2075asGfsgcdCu(G2p)cuu 2199 UUUCUCAGCAUUCAAGCAGGCC 2323 1297616.1gaaUfgCfugagasasa U AD- csuscagcAfuUfCfAfagcaggccuuL96 2076asAfsggdCc(Tgn)gcu 2200 UUCUCAGCAUUCAAGCAGGCCU 2324 1297617.1ugaAfuGfcugagsasa C AD- uscsagcaUfuCfAfAfgcaggccucuL96 2077asGfsagdGc(C2p)ugc 2201 UCUCAGCAUUCAAGCAGGCCUC 2325 1297618.1uugAfaUfgcugasgsa C AD- csasgcauUfcAfAfGfcaggccuccuL96 2078asGfsgadGg(C2p)cug 2202 CUCAGCAUUCAAGCAGGCCUCC 2326 1297619.1cuuGfaAfugcugsasg C AD- asasgaagGfuUfCfCfaggguuuguuL96 2079asAfscaaAfcccuggaA 2203 CUAAGAAGGUUCCAGGGUUUGU 2327 1297620.1fcCfuucuusasg U AD- asgsaaggUfuCfCfAfggguuuguuuL96 2080asAfsacaAfacccuggA 2204 UAAGAAGGUUCCAGGGUUUGUU 2328 1297621.1faCfcuucususa U AD- gsasagguUfcCfAfGfgguuuguuuuL96 2081asAfsaacAfaacccugG 2205 AAGAAGGUUCCAGGGUUUGUUU 2329 1297622.1faAfccuucsusu G AD- asasgguuCfcAfGfGfguuuguuuguL96 2082asCfsaaaCfaaacccuG 2206 AGAAGGUUCCAGGGUUUGUUUG 2330 1297623.1fgAfaccuuscsu U AD- asgsguucCfaGfGfGfuuuguuuguuL96 2083asAfscaaAfcaaacccU 2207 GAAGGUUCCAGGGUUUGUUUGU 2331 1297624.1fgGfaaccususc U AD- gsgsuuccAfgGfGfUfuuguuuguuuL96 2084asAfsacaAfacaaaccC 2208 AAGGUUCCAGGGUUUGUUUGUU 2332 1297625.1fuGfgaaccsusu U AD- gsusuccaGfgGfUfUfuguuuguuuuL96 2085asAfsaacAfaacaaacC 2209 AGGUUCCAGGGUUUGUUUGUUU 2333 1297626.1fcUfggaacscsu C AD- ususccagGfgUfUfUfguuuguuucuL96  812asGfsaaaCfaaacaaaC 1171 GGUUCCAGGGUUUGUUUGUUUC 1530 1136050.2fcCfuggaascsc A AD- uscscaggGfuUfUfGfuuuguuucauL96 2086asUfsgaaAfcaaacaaA 2210 GUUCCAGGGUUUGUUUGUUUCA 2334 1297627.1fcCfcuggasasc U AD- cscsagggUfuUfGfUfuuguuucauuL96 2087asAfsugaAfacaaacaA 2211 UUCCAGGGUUUGUUUGUUUCAU 2335 1297628.1faCfccuggsasa U AD- csasggguUfuGfUfUfuguuucauuuL96  813asAfsaugAfaacaaacA 1172 UCCAGGGUUUGUUUGUUUCAUU 1531 1136051.2faAfcccugsgsa U AD- gsgsguuuGfuUfUfGfuuucauuucuL96  814asGfsaaaUfgaaacaaA 1173 CAGGGUUUGUUUGUUUCAUUUC 1532 1136052.2fcAfaacccsusg C AD- gsgsuuugUfuUfGfUfuucauuuccuL96  815asGfsgaaAfugaaacaA 1174 AGGGUUUGUUUGUUUCAUUUCC 1533 1136053.2faCfaaaccscsu G AD- gsusuuguUfuGfUfUfucauuuccguL96 2088asCfsggaAfaugaaacA 2212 GGGUUUGUUUGUUUCAUUUCCG 2336 1297629.1faAfcaaacscsc C AD- ususuguuUfgUfUfUfcauuuccgcuL96 2089asGfscggAfaaugaaaC 2213 GGUUUGUUUGUUUCAUUUCCGC 2337 1297630.1faAfacaaascsc U AD- ususguuuGfuUfUfCfauuuccgcuuL96  816asAfsgcgGfaaaugaaA 1175 GUUUGUUUGUUUCAUUUCCGCU 1534 1136054.2fcAfaacaasasc G AD- usgsuuugUfuUfCfAfuuuccgcuguL96 2090asCfsagcGfgaaaugaA 2214 UUUGUUUGUUUCAUUUCCGCUG 2338 1297631.1faCfaaacasasa A AD- gsusuuguUfuCfAfUfuuccgcugauL96 2091asUfscadGc(G2p)gaa 2215 UUGUUUGUUUCAUUUCCGCUGA 2339 1297632.1augAfaAfcaaacsasa U AD- ususuguuUfcAfUfUfuccgcugauuL96 2092asAfsucdAg(C2p)gga 2216 UGUUUGUUUCAUUUCCGCUGAU 2340 1297633.1aauGfaAfacaaascsa G AD- ususguuuCfaUfUfUfccgcugauguL96 2093asCfsaudCa(G2p)cgg 2217 GUUUGUUUCAUUUCCGCUGAUG 2341 1297634.1aaaUfgAfaacaasasc A AD- usgsuuucAfuUfUfCfcgcugaugauL96 2094asUfscadTc(Agn)gcg 2218 UUUGUUUCAUUUCCGCUGAUGA 2342 1297635.1gaaAfuGfaaacasasa U AD- gsusuucaUfuUfCfCfgcugaugauuL96 2095asAfsucdAu(C2p)agc 2219 UUGUUUCAUUUCCGCUGAUGAU 2343 1297636.1ggaAfaUfgaaacsasa G AD- ususucauUfuCfCfGfcugaugauguL96 2096asCfsaucAfucagcggA 2220 UGUUUCAUUUCCGCUGAUGAUG 2344 1297637.1faAfugaaascsa U AD- gsgsagauGfgAfGfCfucuuuguguuL96 2097asAfscacAfaagagcuC 2221 GGGGAGAUGGAGCUCUUUGUGU 2345 1297638.1fcAfucuccscsc C AD- gsasgaugGfaGfCfUfcuuugugucuL96 2098asGfsacaCfaaagagcU 2222 GGGAGAUGGAGCUCUUUGUGUC 2346 1297639.1fcCfaucucscsc U AD- asgsauggAfgCfUfCfuuugugucuuL96 2099asAfsgadCa(C2p)aaa 2223 GGAGAUGGAGCUCUUUGUGUCU 2347 1297640.1gagCfuCfcaucuscsc A AD- gsasuggaGfcUfCfUfuugugucuauL96 2100asUfsagdAc(Agn)caa 2224 GAGAUGGAGCUCUUUGUGUCUA 2348 1297641.1agaGfcUfccaucsusc C AD- asusggagCfuCfUfUfugugucuacuL96 2101asGfsuadGa(C2p)aca 2225 AGAUGGAGCUCUUUGUGUCUAC 2349 1297642.1aagAfgCfuccauscsu A AD- usgsgagcUfcUfUfUfgugucuacauL96 2102asUfsgudAg(Agn)cac 2226 GAUGGAGCUCUUUGUGUCUACA 2350 1297643.1aaaGfaGfcuccasusc C AD- gsgsagcuCfuUfUfGfugucuacacuL96 2103asGfsugdTa(G2p)aca 2227 AUGGAGCUCUUUGUGUCUACAC 2351 1297644.1caaAfgAfgcuccsasu A AD- gsasgcucUfuUfGfUfgucuacacauL96 2104asUfsgudGu(Agn)gac 2228 UGGAGCUCUUUGUGUCUACACA 2352 1297645.1acaAfaGfagcucscsa G AD- asgscucuUfuGfUfGfucuacacaguL96  835asCfsugdTg(Tgn)aga 1194 GGAGCUCUUUGUGUCUACACAG 1553 1136073.2cacAfaAfgagcuscsc A AD- gscsucuuUfgUfGfUfcuacacagauL96  836asUfscudGu(G2p)uag 1195 GAGCUCUUUGUGUCUACACAGA 1554 1136074.2acaCfaAfagagcsusc A AD- csuscuuuGfuGfUfCfuacacagaauL96  837asUfsucdTg(Tgn)gua 1196 AGCUCUUUGUGUCUACACAGAA 1555 1136075.2gacAfcAfaagagscsu C AD- uscsuuugUfgUfCfUfacacagaacuL96  838asGfsuudCu(G2p)ugu 1197 GCUCUUUGUGUCUACACAGAAC 1556 1136076.2agaCfaCfaaagasgsc A AD- csusuuguGfuCfUfAfcacagaacauL96  839asUfsgudTc(Tgn)gug 1198 CUCUUUGUGUCUACACAGAACA 1557 1136077.2uagAfcAfcaaagsasg C AD- ususugugUfcUfAfCfacagaacacuL96  840asGfsugdTu(C2p)ugu 1199 UCUUUGUGUCUACACAGAACAC 1558 1136078.2guaGfaCfacaaasgsa C AD- ususguguCfuAfCfAfcagaacaccuL96 2105asGfsgudGu(Tgn)cug 2229 CUUUGUGUCUACACAGAACACC 2353 1297646.1uguAfgAfcacaasasg A AD- usgsugucUfaCfAfCfagaacaccauL96 2106asUfsggdTg(Tgn)ucu 2230 UUUGUGUCUACACAGAACACCA 2354 1297647.1gugUfaGfacacasasa U AD- gsusgucuAfcAfCfAfgaacaccauuL96 2107asAfsugdGu(G2p)uuc 2231 UUGUGUCUACACAGAACACCAU 2355 1297648.1uguGfuAfgacacsasa G AD- usgsucuaCfaCfAfGfaacaccauguL96 2108asCfsaudGg(Tgn)guu 2232 UGUGUCUACACAGAACACCAUG 2356 1297649.1cugUfgUfagacascsa A AD- gsuscuacAfcAfGfAfacaccaugauL96 2109asUfscadTg(G2p)ugu 2233 GUGUCUACACAGAACACCAUGA 2357 1297650.1ucuGfuGfuagacsasc A AD- uscsuacaCfaGfAfAfcaccaugaauL96 2110asUfsucdAu(G2p)gug 2234 UGUCUACACAGAACACCAUGAA 2358 1297651.1uucUfgUfguagascsa G AD- csusacacAfgAfAfCfaccaugaaguL96 2111asCfsuucAfugguguuC 2235 GUCUACACAGAACACCAUGAAG 2359 1297652.1fuGfuguagsasc A AD- usascacaGfaAfCfAfccaugaagauL96 2112asUfscudTc(Agn)ugg 2236 UCUACACAGAACACCAUGAAGA 2360 1297653.1uguUfcUfguguasgsa C AD- gsgsgaacAfcCfCfAfggaucucucuL96 2113asGfsagdAg(Agn)ucc 2237 UGGGGAACACCCAGGAUCUCUC 2361 1297654.1uggGfuGfuucccscsa U AD- gsgsaacaCfcCfAfGfgaucucucuuL96 2114asAfsgadGa(G2p)auc 2238 GGGGAACACCCAGGAUCUCUCU 2362 1297655.1cugGfgUfguuccscsc C AD- gsasacacCfcAfGfGfaucucucucuL96 2115asGfsagdAg(Agn)gau 2239 GGGAACACCCAGGAUCUCUCUC 2363 1297656.1ccuGfgGfuguucscsc A AD- asascaccCfaGfGfAfucucucucauL96 2116asUfsgadGa(G2p)aga 2240 GGAACACCCAGGAUCUCUCUCA 2364 1297657.1uccUfgGfguguuscsc G AD- ascsacccAfgGfAfUfcucucucaguL96 2117asCfsugaGfagagaucC 2241 GAACACCCAGGAUCUCUCUCAG 2365 1297658.1fuGfggugususc A AD- csascccaGfgAfUfCfucucucagauL96 2118asUfscudGa(G2p)aga 2242 AACACCCAGGAUCUCUCUCAGA 2366 1297659.1gauCfcUfgggugsusu G AD- ascsccagGfaUfCfUfcucucagaguL96 2119asCfsucdTg(Agn)gag 2243 ACACCCAGGAUCUCUCUCAGAG 2367 1297660.1agaUfcCfugggusgsu U AD- cscscaggAfuCfUfCfucucagaguuL96 2120asAfscudCu(G2p)aga 2244 CACCCAGGAUCUCUCUCAGAGU 2368 1297661.1gagAfuCfcugggsusg A AD- cscsaggaUfcUfCfUfcucagaguauL96 2121asUfsacdTc(Tgn)gag 2245 ACCCAGGAUCUCUCUCAGAGUA 2369 1297662.1agaGfaUfccuggsgsu U AD- csasggauCfuCfUfCfucagaguauuL96 2122asAfsuadCu(C2p)uga 2246 CCCAGGAUCUCUCUCAGAGUAU 2370 1297663.1gagAfgAfuccugsgsg U AD- asgsgaucUfcUfCfUfcagaguauuuL96  844asAfsaudAc(Tgn)cug 1203 CCAGGAUCUCUCUCAGAGUAUU 1562 1136082.2agaGfaGfauccusgsg A AD- gsgsaucuCfuCfUfCfagaguauuauL96  845asUfsaadTa(C2p)ucu 1204 CAGGAUCUCUCUCAGAGUAUUA 1563 1136083.2gagAfgAfgauccsusg U AD- gsasucucUfcUfCfAfgaguauuauuL96  846asAfsuaaUfacucugaG 1205 AGGAUCUCUCUCAGAGUAUUAU 1564 1136084.2faGfagaucscsu G AD- asuscucuCfuCfAfGfaguauuauguL96  847asCfsauaAfuacucugA 1206 GGAUCUCUCUCAGAGUAUUAUG 1565 1136085.2fgAfgagauscsc G AD- uscsucucUfcAfGfAfguauuaugguL96  848asCfscauAfauacucuG 1207 GAUCUCUCUCAGAGUAUUAUGG 1566 1136086.2faGfagagasusc A AD- csuscucuCfaGfAfGfuauuauggauL96  849asUfsccaUfaauacucU 1208 AUCUCUCUCAGAGUAUUAUGGA 1567 1136087.2fgAfgagagsasu A AD- uscsucucAfgAfGfUfauuauggaauL96  850asUfsuccAfuaauacuC 1209 UCUCUCUCAGAGUAUUAUGGAA 1568 1136088.2fuGfagagasgsa C AD- csuscucaGfaGfUfAfuuauggaacuL96  851asGfsuudCc(Agn)uaa 1210 CUCUCUCAGAGUAUUAUGGAAC 1569 1136089.2uacUfcUfgagagsasg G AD- uscsucagAfgUfAfUfuauggaacguL96  852asCfsguuCfcauaauaC 1211 UCUCUCAGAGUAUUAUGGAACG 1570 1136090.2fuCfugagasgsa A AD- csuscagaGfuAfUfUfauggaacgauL96 2123asUfscgdTu(C2p)cau 2247 CUCUCAGAGUAUUAUGGAACGA 2371 1297664.1aauAfcUfcugagsasg G AD- uscsagagUfaUfUfAfuggaacgaguL96  853asCfsucgUfuccauaaU 1212 UCUCAGAGUAUUAUGGAACGAG 1571 1136091.2faCfucugasgsa C AD- csasgaguAfuUfAfUfggaacgagcuL96  854asGfscucGfuuccauaA 1213 CUCAGAGUAUUAUGGAACGAGC 1572 1136092.2fuAfcucugsasg U AD- asgsaguaUfuAfUfGfgaacgagcuuL96 2124asAfsgcdTc(G2p)uuc 2248 UCAGAGUAUUAUGGAACGAGCU 2372 1297665.1cauAfaUfacucusgsa U AD- gsasguauUfaUfGfGfaacgagcuuuL96 2125asAfsagdCu(C2p)guu 2249 CAGAGUAUUAUGGAACGAGCUU 2373 1297666.1ccaUfaAfuacucsusg U AD- asgsuauuAfuGfGfAfacgagcuuuuL96 2126asAfsaadGc(Tgn)cgu 2250 AGAGUAUUAUGGAACGAGCUUU 2374 1297667.1uccAfuAfauacuscsu A AD- gsusauuaUfgGfAfAfcgagcuuuauL96 2127asUfsaadAg(C2p)ucg 2251 GAGUAUUAUGGAACGAGCUUUA 2375 1297668.1uucCfaUfaauacsusc U AD- usasuuauGfgAfAfCfgagcuuuauuL96 2128asAfsuadAa(G2p)cuc 2252 AGUAUUAUGGAACGAGCUUUAU 2376 1297669.1guuCfcAfuaauascsu U AD- asusuaugGfaAfCfGfagcuuuauuuL96 2129asAfsauaAfagcucguU 2253 GUAUUAUGGAACGAGCUUUAUU 2377 1297670.1fcCfauaausasc C AD- ususauggAfaCfGfAfgcuuuauucuL96 2130asGfsaauAfaagcucgU 2254 UAUUAUGGAACGAGCUUUAUUC 2378 1297671.1fuCfcauaasusa C AD- usasuggaAfcGfAfGfcuuuauuccuL96 2131asGfsgaaUfaaagcucG 2255 AUUAUGGAACGAGCUUUAUUCC 2379 1297672.1fuUfccauasasu A AD- csuscuucCfuGfGfCfugcuucuauuL96 2132asAfsuagAfagcagccA 2256 CCCUCUUCCUGGCUGCUUCUAU 2380 1297673.1fgGfaagagsgsg C AD- uscsuuccUfgGfCfUfgcuucuaucuL96 2133asGfsaudAg(Agn)agc 2257 CCUCUUCCUGGCUGCUUCUAUC 2381 1297674.1agcCfaGfgaagasgsg U AD- csusuccuGfgCfUfGfcuucuaucuuL96 2134asAfsgauAfgaagcagC 2258 CUCUUCCUGGCUGCUUCUAUCU 2382 1297675.1fcAfggaagsasg U AD- ususccugGfcUfGfCfuucuaucuuuL96  920asAfsagaUfagaagcaG 1279 UCUUCCUGGCUGCUUCUAUCUU 1638 1136159.2fcCfaggaasgsa C AD- uscscuggCfuGfCfUfucuaucuucuL96  921asGfsaagAfuagaagcA 1280 CUUCCUGGCUGCUUCUAUCUUC 1639 1136160.2fgCfcaggasasg U AD- cscsuggcUfgCfUfUfcuaucuucuuL96  922asAfsgaaGfauagaagC 1281 UUCCUGGCUGCUUCUAUCUUCU 1640 1136161.2faGfccaggsasa U AD- csusggcuGfcUfUfCfuaucuucuuuL96  923asAfsagaAfgauagaaG 1282 UCCUGGCUGCUUCUAUCUUCUU 1641 1136162.2fcAfgccagsgsa U AD- usgsgcugCfuUfCfUfaucuucuuuuL96  924asAfsaagAfagauagaA 1283 CCUGGCUGCUUCUAUCUUCUUU 1642 1136163.2fgCfagccasgsg G AD- gsgscugcUfuCfUfAfucuucuuuguL96 2135asCfsaaaGfaagauagA 2259 CUGGCUGCUUCUAUCUUCUUUG 2383 1297676.1faGfcagccsasg C AD- gscsugcuUfcUfAfUfcuucuuugcuL96  925asGfscaaAfgaagauaG 1284 UGGCUGCUUCUAUCUUCUUUGC 1643 1136164.2faAfgcagcscsa C AD- csusgcuuCfuAfUfCfuucuuugccuL96  926asGfsgcaAfagaagauA 1285 GGCUGCUUCUAUCUUCUUUGCC 1644 1136165.2fgAfagcagscsc A AD- usgscuucUfaUfCfUfucuuugccauL96  927asUfsggdCa(Agn)aga 1286 GCUGCUUCUAUCUUCUUUGCCA 1645 1136166.2agaUfaGfaagcasgsc U AD- gscsuucuAfuCfUfUfcuuugccauuL96  928asAfsugdGc(Agn)aag 1287 CUGCUUCUAUCUUCUUUGCCAU 1646 1136167.2aagAfuAfgaagcsasg C AD- csusucuaUfcUfUfCfuuugccaucuL96  929asGfsaudGg(C2p)aaa 1288 UGCUUCUAUCUUCUUUGCCAUC 1647 1136168.2gaaGfaUfagaagscsa A AD- ususcuauCfuUfCfUfuugccaucauL96  930asUfsgadTg(G2p)caa 1289 GCUUCUAUCUUCUUUGCCAUCA 1648 1136169.2agaAfgAfuagaasgsc A AD- uscsuaucUfuCfUfUfugccaucaauL96  931asUfsugdAu(G2p)gca 1290 CUUCUAUCUUCUUUGCCAUCAA 1649 1136170.2aagAfaGfauagasasg A AD- csusaucuUfcUfUfUfgccaucaaauL96  932asUfsuudGa(Tgn)ggc 1291 UUCUAUCUUCUUUGCCAUCAAA 1650 1136171.2aaaGfaAfgauagsasa G AD- usasucuuCfuUfUfGfccaucaaaguL96  933asCfsuuuGfauggcaaA 1292 UCUAUCUUCUUUGCCAUCAAAG 1651 1136172.2fgAfagauasgsa A AD- asuscuucUfuUfGfCfcaucaaagauL96  934asUfscudTu(G2p)aug 1293 CUAUCUUCUUUGCCAUCAAAGA 1652 1136173.2gcaAfaGfaagausasg U AD- uscsuucuUfuGfCfCfaucaaagauuL96 2136asAfsucuUfugauggcA 2260 UAUCUUCUUUGCCAUCAAAGAU 2384 1297677.1faAfgaagasusa G AD- csusucuuUfgCfCfAfucaaagauguL96  935asCfsaucUfuugauggC 1294 AUCUUCUUUGCCAUCAAAGAUG 1653 1136174.2faAfagaagsasu C AD- ususcuuuGfcCfAfUfcaaagaugcuL96 2137asGfscadTc(Tgn)uug 2261 UCUUCUUUGCCAUCAAAGAUGC 2385 1297678.1augGfcAfaagaasgsa C AD- uscsuuugCfcAfUfCfaaagaugccuL96 2138asGfsgcdAu(C2p)uuu 2262 CUUCUUUGCCAUCAAAGAUGCC 2386 1297679.1gauGfgCfaaagasasg A AD- csusuugcCfaUfCfAfaagaugccauL96 2139asUfsggdCa(Tgn)cuu 2263 UUCUUUGCCAUCAAAGAUGCCA 2387 1297680.1ugaUfgGfcaaagsasa U AD- ususugccAfuCfAfAfagaugccauuL96 2140asAfsugdGc(Agn)ucu 2264 UCUUUGCCAUCAAAGAUGCCAU 2388 1297681.1uugAfuGfgcaaasgsa C AD- ususgccaUfcAfAfAfgaugccaucuL96 2141asGfsaudGg(C2p)auc 2265 CUUUGCCAUCAAAGAUGCCAUC 2389 1297682.1uuuGfaUfggcaasasg C AD- usgsccauCfaAfAfGfaugccauccuL96 2142asGfsgadTg(G2p)cau 2266 UUUGCCAUCAAAGAUGCCAUCC 2390 1297683.1cuuUfgAfuggcasasa G AD- gscscaucAfaAfGfAfugccauccguL96 2143asCfsggaUfggcaucuU 2267 UUGCCAUCAAAGAUGCCAUCCG 2391 1297684.1fuGfauggcsasa U AD- asasaccaAfuGfAfAfcagcaaagcuL96 2144asGfscudTu(G2p)cug 2268 UCAAACCAAUGAACAGCAAAGC 2392 1297685.1uucAfuUfgguuusgsa A AD- asasccaaUfgAfAfCfagcaaagcauL96 2145asUfsgcdTu(Tgn)gcu 2269 CAAACCAAUGAACAGCAAAGCA 2393 1297686.1guuCfaUfugguususg U AD- ascscaauGfaAfCfAfgcaaagcauuL96 2146asAfsugdCu(Tgn)ugc 2270 AAACCAAUGAACAGCAAAGCAU 2394 1297687.1uguUfcAfuuggususu A AD- cscsaaugAfaCfAfGfcaaagcauauL96 2147asUfsaudGc(Tgn)uug 2271 AACCAAUGAACAGCAAAGCAUA 2395 1297688.1cugUfuCfauuggsusu A AD- csasaugaAfcAfGfCfaaagcauaauL96 2148asUfsuadTg(C2p)uuu 2272 ACCAAUGAACAGCAAAGCAUAA 2396 1297689.1gcuGfuUfcauugsgsu C AD- asasugaaCfaGfCfAfaagcauaacuL96 2149asGfsuudAu(G2p)cuu 2273 CCAAUGAACAGCAAAGCAUAAC 2397 1297690.1ugcUfgUfucauusgsg C AD- asusgaacAfgCfAfAfagcauaaccuL96 2150asGfsgudTa(Tgn)gcu 2274 CAAUGAACAGCAAAGCAUAACC 2398 1297691.1uugCfuGfuucaususg U AD- usgsaacaGfcAfAfAfgcauaaccuuL96 2151asAfsgguUfaugcuuuG 2275 AAUGAACAGCAAAGCAUAACCU 2399 1297692.1fcUfguucasusu U AD- gsasacagCfaAfAfGfcauaaccuuuL96  982asAfsaggUfuaugcuuU 1341 AUGAACAGCAAAGCAUAACCUU 1700 1136221.2fgCfuguucsasu G AD- asascagcAfaAfGfCfauaaccuuguL96  983asCfsaadGg(Tgn)uau 1342 UGAACAGCAAAGCAUAACCUUG 1701 1136222.2gcuUfuGfcuguuscsa A AD- ascsagcaAfaGfCfAfuaaccuugauL96 2152asUfscadAg(G2p)uua 2276 GAACAGCAAAGCAUAACCUUGA 2400 1297693.1ugcUfuUfgcugususc A AD- csasgcaaAfgCfAfUfaaccuugaauL96 2153asUfsucdAa(G2p)guu 2277 AACAGCAAAGCAUAACCUUGAA 2401 1297694.1augCfuUfugcugsusu U AD- asgscaaaGfcAfUfAfaccuugaauuL96  984asAfsuucAfagguuauG 1343 ACAGCAAAGCAUAACCUUGAAU 1702 1136223.2fcUfuugcusgsu C AD- gscsaaagCfaUfAfAfccuugaaucuL96  985asGfsaudTc(Agn)agg 1344 CAGCAAAGCAUAACCUUGAAUC 1703 1136224.2uuaUfgCfuuugcsusg U AD- csasaagcAfuAfAfCfcuugaaucuuL96  986asAfsgadTu(C2p)aag 1345 AGCAAAGCAUAACCUUGAAUCU 1704 1136225.2guuAfuGfcuuugscsu A AD- asasagcaUfaAfCfCfuugaaucuauL96 2154asUfsagdAu(Tgn)caa 2278 GCAAAGCAUAACCUUGAAUCUA 2402 1297695.1gguUfaUfgcuuusgsc U AD- asasgcauAfaCfCfUfugaaucuauuL96 2155asAfsuagAfuucaaggU 2279 CAAAGCAUAACCUUGAAUCUAU 2403 1297696.1fuAfugcuususg A AD- asgscauaAfcCfUfUfgaaucuauauL96 2156asUfsaudAg(Agn)uuc 2280 AAAGCAUAACCUUGAAUCUAUA 2404 1297697.1aagGfuUfaugcususu C AD- gscsauaaCfcUfUfGfaaucuauacuL96  987asGfsuauAfgauucaaG 1346 AAGCAUAACCUUGAAUCUAUAC 1705 1136226.2fgUfuaugcsusu U AD- csasuaacCfuUfGfAfaucuauacuuL96  988asAfsguaUfagauucaA 1347 AGCAUAACCUUGAAUCUAUACU 1706 1136227.2fgGfuuaugscsu C AD- asusaaccUfuGfAfAfucuauacucuL96  989asGfsaguAfuagauucA 1348 GCAUAACCUUGAAUCUAUACUC 1707 1136228.2faGfguuausgsc A AD- usasaccuUfgAfAfUfcuauacucauL96  990asUfsgadGu(Agn)uag 1349 CAUAACCUUGAAUCUAUACUCA 1708 1136229.2auuCfaAfgguuasusg A AD- asasccuuGfaAfUfCfuauacucaauL96  991asUfsugdAg(Tgn)aua 1350 AUAACCUUGAAUCUAUACUCAA 1709 1136230.2gauUfcAfagguusasu A AD- ascscuugAfaUfCfUfauacucaaauL96  992asUfsuudGa(G2p)uau 1351 UAACCUUGAAUCUAUACUCAAA 1710 1136231.2agaUfuCfaaggususa U AD- cscsuugaAfuCfUfAfuacucaaauuL96  993asAfsuudTg(Agn)gua 1352 AACCUUGAAUCUAUACUCAAAU 1711 1136232.2uagAfuUfcaaggsusu U AD- csusugaaUfcUfAfUfacucaaauuuL96 2157asAfsaudTu(G2p)agu 2281 ACCUUGAAUCUAUACUCAAAUU 2405 1297698.1auaGfaUfucaagsgsu U AD- ususgaauCfuAfUfAfcucaaauuuuL96 2158asAfsaauUfugaguauA 2282 CCUUGAAUCUAUACUCAAAUUU 2406 1297699.1fgAfuucaasgsg U AD- gsasaucuAfuAfCfUfcaaauuuuguL96 2159asCfsaaaAfuuugaguA 2283 UUGAAUCUAUACUCAAAUUUUG 2407 1297700.1fuAfgauucsasa C AD- asasucuaUfaCfUfCfaaauuuugcuL96  994asGfscaaAfauuugagU 1353 UGAAUCUAUACUCAAAUUUUGC 1712 1136233.2faUfagauuscsa A AD- asuscuauAfcUfCfAfaauuuugcauL96  995asUfsgcaAfaauuugaG 1354 GAAUCUAUACUCAAAUUUUGCA 1713 1136234.2fuAfuagaususc A AD- uscsuauaCfuCfAfAfauuuugcaauL96 2160asUfsugcAfaaauuugA 2284 AAUCUAUACUCAAAUUUUGCAA 2408 1297701.1fgUfauagasusu U AD- csusauacUfcAfAfAfuuuugcaauuL96 2161asAfsuudGc(Agn)aaa 2285 AUCUAUACUCAAAUUUUGCAAU 2409 1297702.1uuuGfaGfuauagsasu G AD- usasuacuCfaAfAfUfuuugcaauguL96 2162asCfsaudTg(C2p)aaa 2286 UCUAUACUCAAAUUUUGCAAUG 2410 1297703.1auuUfgAfguauasgsa A AD- asusacucAfaAfUfUfuugcaaugauL96 2163asUfscadTu(G2p)caa 2287 CUAUACUCAAAUUUUGCAAUGA 2411 1297704.1aauUfuGfaguausasg G AD- usascucaAfaUfUfUfugcaaugaguL96 2164asCfsucaUfugcaaaaU 2288 UAUACUCAAAUUUUGCAAUGAG 2412 1297705.1fuUfgaguasusa G AD- ascsucaaAfuUfUfUfgcaaugagguL96 2165asCfscucAfuugcaaaA 2289 AUACUCAAAUUUUGCAAUGAGG 2413 1297706.1fuUfugagusasu C AD- csuscaaaUfuUfUfGfcaaugaggcuL96 2166asGfsccdTc(Agn)uug 2290 UACUCAAAUUUUGCAAUGAGGC 2414 1297707.1caaAfaUfuugagsusa A AD- uscsaaauUfuUfGfCfaaugaggcauL96 2167asUfsgcdCu(C2p)auu 2291 ACUCAAAUUUUGCAAUGAGGCA 2415 1297708.1gcaAfaAfuuugasgsu G AD- csasaauuUfuGfCfAfaugaggcaguL96 2168asCfsugdCc(Tgn)cau 2292 CUCAAAUUUUGCAAUGAGGCAG 2416 1297709.1ugcAfaAfauuugsasg U AD- asasauuuUfgCfAfAfugaggcaguuL96 2169asAfscudGc(C2p)uca 2293 UCAAAUUUUGCAAUGAGGCAGU 2417 1297710.1uugCfaAfaauuusgsa G AD- asasuuuuGfcAfAfUfgaggcaguguL96 2170asCfsacdTg(C2p)cuc 2294 CAAAUUUUGCAAUGAGGCAGUG 2418 1297711.1auuGfcAfaaauususg G AD- asusuuugCfaAfUfGfaggcagugguL96 2171asCfscacUfgccucauU 2295 AAAUUUUGCAAUGAGGCAGUGG 2419 1297712.1fgCfaaaaususu G AD- csuscuccaagUfAfUfgaucgucuuL96 2172asdAsgadCgdAucaudA 2296 UGCUCUCCAAGUAUGAUCGUCU 1476 1395794.1cUfuggagagscsg G AD- asgsgaucucUfCfUfcagaguauuuL96 2173asdAsaudAc(Tgn)cug 2297 CCAGGAUCUCUCUCAGAGUAUU 1562 1395797.1adGadGadGauccusgsg A AD- csasgugaugCfUfCfuccaaguauuL96 2174asdAsuadCudTggagdA 2298 CACAGUGAUGCUCUCCAAGUAU 2305 1395803.1gCfaucacugsusg G AD- cscsaaguauGfAfUfcgucugcaguL96 2175asdCsugdCadGacgadT 2299 CUCCAAGUAUGAUCGUCUGCAG 2312 1395805.1cAfuacuuggsasg A AD- gsusuuguuuCfAfUfuuccgcugauL96 2176asdTscadGc(G2p)gaa 2300 UUGUUUGUUUCAUUUCCGCUGA 2339 1395807.1adTgAfaacaaacsgsg U AD- asascacccaGfGfAfucucucucauL96 2177asdTsgadGa(G2p)aga 2301 GGAACACCCAGGAUCUCUCUCA 2364 1395811.1udCcUfggguguuscsc G AD- gsgsguuuguUfudGUfuucauuucuL96 2178asdGsaadAudGaaacdA 2302 CAGGGUUUGUUUGUUUCAUUUC 1532 1395816.1aAfcaaacccsusg C AD- gsasguauuuCfUfCfagcauucaauL96 2179asdTsugdAadTgcugdA 2303 GGGAGUAUUUCUCAGCAUUCAA 1519 1395823.1gAfaauacucscsc G

TABLE 8 Xanthine Dehydrogenase dsRNA Agent In Vitro Single Dose Screensin Primary Human Hepatocytes PHH 500 nM 100 nM 10 nM 0.1 nM % of Avg %of Avg % of Avg % of Avg Message ST Message ST Message ST Message STDuplexID Remaining DEV Remaining DEV Remaining DEV Remaining DEVAD-1135990.2 69.5 32.5 49.2 13.7 87.8 12.8 68.7 9.1 AD-1135991.2 36.55.0 47.2 4.8 73.1 16.7 85.6 18.1 AD-1297597.1 34.7 7.4 36.6 15.2 80.023.2 76.5 5.6 AD-1297598.1 29.7 8.7 37.2 5.8 65.1 13.4 51.9 27.4AD-1297599.1 47.9 15.5 59.7 6.3 110.0 22.3 74.3 23.6 AD-1297600.1 37.711.6 60.6 5.6 87.2 4.5 67.7 15.9 AD-1297601.1 43.3 7.9 61.2 19.4 91.811.2 93.9 12.0 AD-1135992.2 34.0 6.5 31.7 16.9 64.5 31.3 90.8 26.9AD-1135993.2 54.4 5.9 60.8 13.9 107.4 31.0 111.3 5.6 AD-1135994.2 84.88.6 95.6 8.2 109.1 15.8 119.7 11.0 AD-1135995.2 59.0 4.3 75.2 14.3 94.926.1 95.8 15.0 AD-1135996.2 31.1 4.3 50.8 7.1 63.5 16.7 80.2 21.7AD-1297602.1 74.7 7.3 73.2 12.7 104.5 7.3 84.3 21.0 AD-1297603.1 91.49.7 142.7 74.0 117.3 17.7 110.9 14.0 AD-1297604.1 50.1 10.9 57.4 8.895.0 29.8 120.8 28.8 AD-1297605.1 30.8 4.7 35.7 25.5 86.0 26.2 140.833.5 AD-1297606.1 86.7 18.6 68.4 12.6 85.1 7.2 89.8 6.6 AD-1297607.147.3 3.0 56.6 18.7 70.1 7.6 103.3 8.5 AD-1297608.1 47.2 7.7 49.6 10.565.7 9.5 84.7 20.3 AD-1297609.1 63.2 6.4 61.0 7.5 96.6 16.7 112.8 22.7AD-1297610.1 60.3 9.7 78.9 10.5 104.7 12.3 118.0 17.4 AD-1297611.1 65.714.9 70.0 9.6 88.0 10.6 116.1 13.8 AD-1136037.2 35.8 3.7 27.7 7.9 78.213.9 138.3 0.0 AD-1136038.2 37.6 1.1 26.3 10.8 77.8 8.7 86.1 27.4AD-1136039.2 42.2 7.7 75.4 8.1 88.3 9.9 96.2 22.8 AD-1136040.2 63.0 9.889.8 7.8 86.4 21.0 89.9 11.6 AD-1136041.2 83.0 16.4 70.0 21.5 105.9 21.498.7 5.0 AD-1297612.1 84.2 17.1 83.6 5.9 106.2 14.6 103.2 7.8AD-1297613.1 52.7 20.3 89.6 28.1 97.3 15.1 85.6 12.9 AD-1297614.1 72.614.9 70.6 30.7 74.5 17.3 96.5 12.5 AD-1297615.1 73.9 6.0 94.3 31.6 96.210.0 113.8 37.2 AD-1297616.1 69.5 12.7 89.8 4.4 103.9 23.4 102.4 31.5AD-1297617.1 62.5 17.7 53.4 8.0 79.5 9.8 87.2 5.8 AD-1297618.1 48.2 0.076.0 12.7 106.7 35.5 81.6 10.8 AD-1297619.1 90.3 21.9 81.4 9.4 106.314.9 96.6 24.9 AD-1297620.1 48.1 11.5 47.7 4.6 90.3 28.9 111.2 26.6AD-1297621.1 43.9 12.6 54.0 12.5 88.6 18.3 84.4 20.6 AD-1297622.1 52.217.4 65.1 23.1 103.5 8.3 105.1 25.5 AD-1297623.1 79.5 3.9 78.2 21.8107.4 6.1 107.4 14.9 AD-1297624.1 46.9 22.9 66.8 22.5 94.6 10.2 102.224.4 AD-1297625.1 71.6 25.9 88.4 16.0 84.4 34.9 110.2 31.1 AD-1297626.174.1 21.9 61.7 12.1 89.4 14.6 98.4 13.2 AD-1136050.2 47.9 27.7 62.8 27.389.2 16.2 102.8 34.7 AD-1297627.1 42.7 21.0 43.9 10.8 90.9 28.5 112.429.7 AD-1297628.1 27.2 2.8 40.3 11.7 63.8 22.0 73.5 28.0 AD-1136051.237.4 5.3 44.9 12.0 45.0 11.8 91.8 11.6 AD-1136052.2 41.2 10.0 44.4 11.276.3 4.5 70.5 28.6 AD-1136053.2 38.7 7.0 68.6 8.4 82.4 38.3 129.7 35.5AD-1297629.1 53.8 17.9 57.9 11.7 89.1 15.3 137.6 34.8 AD-1297630.1 58.723.3 85.6 17.4 96.6 21.8 70.5 33.0 AD-1136054.2 53.3 22.5 62.8 10.3 85.725.7 107.7 24.2 AD-1297631.1 48.2 15.8 80.4 15.6 107.1 16.3 110.5 25.1AD-1297632.1 42.1 7.2 43.0 10.0 96.2 9.7 103.3 1.0 AD-1297633.1 43.2 8.990.4 11.0 96.5 20.0 93.0 6.0 AD-1297634.1 54.6 15.4 88.7 21.6 85.9 16.7106.6 12.6 AD-1297635.1 67.7 14.5 74.6 16.9 102.2 24.8 84.7 26.6AD-1297636.1 80.8 15.9 93.2 14.2 106.6 23.4 120.7 34.7 AD-1297637.1 40.74.8 73.3 18.4 72.2 11.8 105.2 17.1 AD-1297638.1 79.9 14.7 98.6 15.9120.9 20.9 97.7 31.5 AD-1297639.1 41.6 9.7 84.5 15.6 107.2 11.6 128.29.5 AD-1297640.1 82.8 18.5 82.7 23.1 97.3 15.4 199.9 12.3 AD-1297641.174.2 6.0 90.5 31.3 92.7 6.3 101.5 20.7 AD-1297642.1 50.8 13.4 87.0 13.382.3 6.7 101.5 15.8 AD-1297643.1 44.1 6.1 79.7 n/a 102.6 15.2 120.1 24.2AD-1297644.1 73.8 19.0 87.5 23.9 80.4 22.3 99.8 36.1 AD-1297645.1 80.826.6 100.5 15.6 92.8 31.6 103.2 15.6 AD-1136073.2 54.6 20.8 125.2 25.4135.6 21.5 114.8 14.6 AD-1136074.2 72.1 n/a 79.6 7.4 88.6 19.7 129.9 3.6AD-1136075.2 38.2 9.0 52.3 15.5 90.8 28.0 76.2 15.0 AD-1136076.2 82.615.6 114.4 55.2 116.3 29.6 116.6 26.6 AD-1136077.2 58.6 19.5 72.0 8.482.5 19.2 102.0 33.4 AD-1136078.2 86.7 5.2 129.5 18.3 118.8 14.8 108.619.1 AD-1297646.1 69.8 17.8 123.8 58.6 93.4 10.9 137.9 28.8 AD-1297647.158.3 9.4 56.0 16.9 122.6 18.1 119.8 18.6 AD-1297648.1 86.3 12.5 90.3 0.095.0 23.8 140.1 35.6 AD-1297649.1 42.0 13.5 70.2 14.8 92.5 15.1 90.3 4.5AD-1297650.1 68.8 6.7 80.5 18.9 103.1 8.2 121.7 8.8 AD-1297651.1 68.35.8 99.0 28.3 73.4 14.4 105.5 22.0 AD-1297652.1 99.0 30.9 109.3 18.4111.7 25.7 103.6 8.6 AD-1297653.1 53.3 15.2 88.9 62.0 111.2 27.5 95.216.1 AD-1297654.1 126.3 16.6 110.5 19.0 105.7 10.3 91.4 8.1 AD-1297655.1116.3 11.7 124.4 22.3 87.0 8.5 126.4 11.7 AD-1297656.1 83.3 14.8 65.26.7 101.1 18.3 90.7 18.0 AD-1297657.1 22.0 8.2 51.6 49.2 62.9 8.9 107.530.9 AD-1297658.1 71.1 3.5 78.2 14.6 103.9 10.2 106.4 11.4 AD-1297659.163.9 18.4 73.0 22.4 89.1 10.1 95.7 18.8 AD-1297660.1 54.6 13.9 70.9 44.2101.9 19.1 90.4 19.2 AD-1297661.1 67.9 35.5 61.7 14.7 91.0 23.6 81.033.3 AD-1297662.1 74.3 15.3 69.9 16.7 97.1 21.9 79.0 19.9 AD-1297663.177.0 24.1 36.6 7.8 85.9 25.6 103.4 7.0 AD-1136082.2 57.0 0.6 65.1 13.347.7 8.6 n/d n/d AD-1136083.2 82.2 8.9 76.8 26.0 41.1 19.0 n/d n/dAD-1136084.2 81.8 7.2 87.4 13.5 50.2 8.6 n/d n/d AD-1136085.2 98.6 21.4102.9 38.2 75.8 30.0 n/d n/d AD-1136086.2 91.7 33.5 102.1 10.8 67.5 8.4n/d n/d AD-1136087.2 87.2 23.0 63.5 1.9 54.8 12.9 n/d n/d AD-1136088.299.5 11.4 73.3 24.0 69.1 7.2 n/d n/d AD-1136089.2 87.8 11.4 96.4 43.357.6 18.6 n/d n/d AD-1136090.2 101.3 15.8 116.1 14.8 74.6 20.4 n/d n/dAD-1297664.1 73.8 12.3 84.0 6.5 65.7 3.8 n/d n/d AD-1136091.2 52.3 14.558.2 11.3 55.3 15.3 n/d n/d AD-1136092.2 62.4 6.9 71.3 10.6 55.6 26.6n/d n/d AD-1297665.1 62.9 10.6 75.5 6.9 62.1 11.5 n/d n/d AD-1297666.154.8 13.8 59.6 15.2 51.1 7.3 n/d n/d AD-1297667.1 65.8 5.0 66.6 24.963.3 14.4 n/d n/d AD-1297668.1 66.2 10.2 68.8 3.7 54.6 15.8 n/d n/dAD-1297669.1 73.4 9.4 72.2 2.5 65.9 13.6 n/d n/d AD-1297670.1 57.1 7.373.4 19.6 61.2 8.5 n/d n/d AD-1297671.1 63.1 13.1 94.9 24.4 88.0 6.3 n/dn/d AD-1297672.1 123.7 12.5 124.9 10.9 101.4 27.4 n/d n/d AD-1297673.160.0 10.8 66.5 4.4 71.3 16.0 n/d n/d AD-1297674.1 96.2 26.1 111.6 8.5110.5 9.5 n/d n/d AD-1297675.1 75.1 3.9 95.9 3.3 73.9 48.8 n/d n/dAD-1136159.2 56.0 6.6 106.5 19.2 55.0 9.8 n/d n/d AD-1136160.2 69.8 15.384.4 6.2 81.7 8.5 n/d n/d AD-1136161.2 75.8 24.6 96.5 20.6 93.1 5.0 n/dn/d AD-1136162.2 73.6 8.4 74.9 19.7 76.2 10.7 n/d n/d AD-1136163.2 62.414.7 86.2 18.7 55.2 20.3 n/d n/d AD-1297676.1 59.9 11.9 65.8 22.6 88.136.6 n/d n/d AD-1136164.2 94.5 11.4 100.5 14.2 92.6 23.3 n/d n/dAD-1136165.2 69.2 16.2 71.0 6.2 64.7 9.2 n/d n/d AD-1136166.2 47.1 9.856.5 21.4 70.8 5.0 n/d n/d AD-1136167.2 107.0 14.0 102.6 27.8 86.2 9.9n/d n/d AD-1136168.2 104.9 22.3 86.7 26.5 98.1 15.2 n/d n/d AD-1136169.264.4 21.6 54.5 16.6 67.6 15.3 n/d n/d AD-1136170.2 64.6 14.6 66.7 12.080.8 12.9 n/d n/d AD-1136171.2 100.8 17.6 111.3 13.2 106.0 21.1 n/d n/dAD-1136172.2 72.0 14.2 88.1 23.7 69.4 28.7 n/d n/d AD-1136173.2 97.426.7 103.0 57.7 78.0 1.1 n/d n/d AD-1297677.1 96.1 26.8 106.2 38.5 93.426.2 n/d n/d AD-1136174.2 75.2 20.0 96.4 10.3 100.4 10.2 n/d n/dAD-1297678.1 118.3 19.4 111.5 20.6 103.2 15.4 n/d n/d AD-1297679.1 116.012.2 112.6 31.9 111.8 7.4 n/d n/d AD-1297680.1 90.0 27.9 95.8 29.6 105.820.4 n/d n/d AD-1297681.1 105.9 13.9 109.2 10.6 111.6 16.4 n/d n/dAD-1297682.1 77.2 14.6 92.4 4.5 76.6 16.7 n/d n/d AD-1297683.1 64.0 14.890.5 16.1 71.8 14.7 n/d n/d AD-1297684.1 87.4 2.0 87.7 15.4 71.7 31.3n/d n/d AD-1297685.1 90.2 15.1 94.3 5.2 113.9 38.3 n/d n/d AD-1297686.194.7 9.8 80.8 7.5 117.1 21.2 n/d n/d AD-1297687.1 58.8 11.8 79.6 2.4106.6 18.3 n/d n/d AD-1297688.1 82.2 5.0 78.0 26.0 78.6 6.3 n/d n/dAD-1297689.1 59.7 21.1 86.7 1.7 74.8 25.1 n/d n/d AD-1297690.1 74.9 26.286.5 11.0 74.1 25.1 n/d n/d AD-1297691.1 88.5 22.1 103.7 14.2 90.9 18.6n/d n/d AD-1297692.1 105.8 21.4 103.4 26.9 86.1 8.3 n/d n/d AD-1136221.267.9 32.7 72.6 12.3 100.3 10.7 n/d n/d AD-1136222.2 94.3 12.7 127.7 28.693.0 4.6 n/d n/d AD-1297693.1 77.2 25.7 97.6 22.6 113.9 12.6 n/d n/dAD-1297694.1 81.4 10.1 109.5 5.5 94.1 9.6 n/d n/d AD-1136223.2 64.0 8.461.8 21.1 98.3 20.4 n/d n/d AD-1136224.2 63.4 2.3 62.9 15.0 67.7 25.1n/d n/d AD-1136225.2 87.1 19.2 92.7 11.0 63.9 14.1 n/d n/d AD-1297695.180.2 16.5 77.9 3.4 100.9 21.4 n/d n/d AD-1297696.1 71.7 12.4 70.7 21.090.1 16.0 n/d n/d AD-1297697.1 108.1 5.3 89.0 12.7 119.6 13.2 n/d n/dAD-1136226.2 92.0 4.1 95.4 29.2 100.4 14.5 n/d n/d AD-1136227.2 60.515.7 91.0 1.8 74.5 17.4 n/d n/d AD-1136228.2 61.2 4.0 77.8 18.6 57.013.0 n/d n/d AD-1136229.2 78.3 12.0 80.1 8.3 89.1 18.7 n/d n/dAD-1136230.2 92.2 19.3 106.9 14.0 82.9 14.7 n/d n/d AD-1136231.2 96.912.5 85.5 14.0 95.5 14.0 n/d n/d AD-1136232.2 79.5 8.3 73.1 24.7 87.615.3 n/d n/d AD-1297698.1 76.3 7.0 79.1 23.3 101.6 21.5 n/d n/dAD-1297699.1 44.5 12.0 67.5 17.5 87.0 19.1 n/d n/d AD-1297700.1 86.319.6 82.6 19.5 58.2 8.5 n/d n/d AD-1136233.2 86.6 14.4 80.5 9.5 73.611.6 n/d n/d AD-1136234.2 85.4 15.2 77.1 13.1 80.3 17.2 n/d n/dAD-1297701.1 79.3 8.4 84.4 14.6 72.0 24.3 n/d n/d AD-1297702.1 100.3 3.283.1 11.3 95.1 14.0 n/d n/d AD-1297703.1 78.0 26.3 117.3 16.3 112.4 34.5n/d n/d AD-1297704.1 85.1 8.4 95.1 24.5 111.8 37.5 n/d n/d AD-1297705.174.4 17.9 84.1 56.6 103.6 6.6 n/d n/d AD-1297706.1 101.6 28.1 68.8 32.447.8 6.1 n/d n/d AD-1297707.1 94.1 21.1 86.9 8.5 62.1 23.6 n/d n/dAD-1297708.1 80.7 18.7 76.0 12.5 82.0 13.4 n/d n/d AD-1297709.1 78.1 8.874.3 26.0 72.2 18.5 n/d n/d AD-1297710.1 68.9 12.3 79.7 20.2 79.0 14.7n/d n/d AD-1297711.1 78.2 17.6 95.3 27.3 72.0 9.0 n/d n/d AD-1297712.186.2 10.5 88.8 16.4 90.9 26.1 n/d n/d

TABLE 9 Xanthine Dehydrogenase dsRNA Agent In Vitro Single Dose Screensin Primary Human Hepatocytes PHH 500 nM 100 nM 10 nM 0.1 nM % of Avg %of Avg % of Avg % of Avg Message ST Message ST Message ST Message STDuplexID Remaining DEV Remaining DEV Remaining DEV Remaining DEVAD-1135990.2 77.7 14.6 46.5 24.5 40.3 26.2 53.9 33.2 AD-1135991.2 60.715.3 35.9 16.3 41.1 14.3 75.1 36.6 AD-1297597.1 56.8 25.9 36.9 11.8 72.045.0 51.9 24.4 AD-1297598.1 44.7 28.8 69.4 28.7 48.5 26.4 79.8 25.9AD-1297599.1 83.4 29.1 97.6 37.6 50.7 15.3 78.5 20.8 AD-1297600.1 88.121.6 43.5 12.7 45.6 7.3 70.4 21.5 AD-1297601.1 57.5 40.4 46.3 11.3 49.59.4 72.6 21.3 AD-1135992.2 50.9 17.5 43.7 6.5 33.5 7.4 38.5 25.0AD-1135993.2 58.0 16.6 79.5 21.3 63.4 48.2 71.4 39.9 AD-1135994.2 117.814.1 81.7 22.1 103.8 30.2 102.9 39.5 AD-1135995.2 131.8 27.0 78.5 22.8152.4 18.4 120.4 28.1 AD-1135996.2 47.0 16.3 49.9 19.2 115.5 18.3 89.322.1 AD-1297602.1 48.7 31.0 106.6 39.7 97.9 27.4 82.2 16.9 AD-1297603.1108.1 42.1 88.7 18.4 81.5 35.9 72.8 14.8 AD-1297604.1 62.9 36.3 66.116.0 69.7 15.9 90.1 9.7 AD-1297605.1 49.1 14.5 45.4 14.3 69.5 44.7 48.916.4 AD-1297606.1 74.2 20.6 79.3 13.2 43.3 7.8 63.1 7.3 AD-1297607.168.6 35.0 63.8 30.0 44.3 10.3 99.1 19.8 AD-1297608.1 75.7 39.0 85.4 38.085.7 21.5 87.4 11.7 AD-1297609.1 114.4 26.2 99.9 35.8 98.8 16.2 95.834.8 AD-1297610.1 87.7 8.0 73.1 31.8 72.3 22.7 102.1 23.6 AD-1297611.193.5 18.0 71.8 20.5 57.3 26.1 81.0 15.2 AD-1136037.2 53.2 8.0 48.1 22.965.4 8.6 52.6 22.2 AD-1136038.2 58.1 25.6 26.4 3.6 37.2 13.9 67.7 21.8AD-1136039.2 72.8 19.0 54.2 30.3 75.6 28.9 82.3 30.9 AD-1136040.2 122.944.6 123.6 56.7 89.2 31.0 67.6 8.7 AD-1136041.2 123.5 38.0 138.3 31.785.7 28.1 131.5 22.0 AD-1297612.1 132.7 41.5 137.2 41.0 107.2 34.9 123.017.7 AD-1297613.1 73.8 31.3 88.3 31.9 83.8 24.7 98.0 22.7 AD-1297614.179.8 12.9 103.4 36.9 67.2 N/A 68.9 30.2 AD-1297615.1 123.9 44.3 94.269.5 86.7 40.1 96.3 43.2 AD-1297616.1 106.0 35.0 85.7 41.7 84.4 29.8112.5 12.9 AD-1297617.1 60.7 22.5 99.8 66.2 85.9 46.1 141.1 32.9AD-1297618.1 111.7 43.5 135.2 44.2 98.6 46.9 109.9 21.0 AD-1297619.1110.0 14.8 133.3 29.2 126.3 42.2 120.9 39.1 AD-1297620.1 84.4 26.6 135.339.8 91.7 15.7 114.7 15.3 AD-1297621.1 60.2 20.8 77.7 7.4 88.2 32.9 85.415.5 AD-1297622.1 77.1 19.3 96.8 29.0 105.2 55.1 69.2 37.5 AD-1297623.185.5 40.2 82.6 28.3 148.1 48.2 92.7 42.3 AD-1297624.1 44.0 5.7 61.2 9.091.6 1.6 104.6 69.2 AD-1297625.1 117.2 42.9 121.1 41.8 131.3 43.9 120.647.1 AD-1297626.1 112.4 26.9 118.6 66.3 134.0 24.6 145.2 41.0AD-1136050.2 62.9 27.3 74.8 13.0 149.8 50.5 142.6 26.7 AD-1297627.1 95.439.7 79.4 26.9 110.3 32.9 129.4 16.4 AD-1297628.1 79.7 27.1 59.1 15.568.3 19.3 96.1 16.2 AD-1136051.2 39.1 21.5 34.8 18.9 53.6 49.8 62.8 16.7AD-1136052.2 77.1 17.9 65.1 44.0 95.6 49.0 98.3 36.1 AD-1136053.2 76.240.2 100.9 6.9 108.5 44.0 101.0 11.9 AD-1297629.1 90.8 46.5 97.0 23.5108.3 33.9 122.0 41.3 AD-1297630.1 119.0 29.3 141.6 67.2 151.1 46.4138.4 32.1 AD-1136054.2 123.4 8.4 104.4 32.9 117.1 27.4 113.9 15.5AD-1297631.1 78.3 9.5 118.5 49.3 127.7 20.5 116.4 36.1 AD-1297632.1 53.110.2 35.8 9.4 93.9 17.2 59.1 19.8 AD-1297633.1 80.5 31.1 68.3 50.4 102.561.9 91.1 8.7 AD-1297634.1 107.7 25.2 101.7 40.4 112.3 18.7 137.5 36.5AD-1297635.1 126.7 22.6 138.4 77.3 142.0 40.9 132.4 15.3 AD-1297636.1126.6 23.1 169.9 18.8 157.9 51.9 143.2 13.6 AD-1297637.1 92.1 39.5 166.855.2 131.7 39.1 134.5 44.5 AD-1297638.1 148.5 15.3 131.7 64.4 142.3 44.7127.2 11.1 AD-1297639.1 106.8 43.2 115.6 12.7 152.9 32.2 112.3 14.5AD-1297640.1 95.4 33.6 74.2 35.4 80.9 39.9 65.6 24.0 AD-1297641.1 62.640.2 75.2 38.8 101.0 40.2 91.7 35.7 AD-1297642.1 94.0 27.1 87.7 25.2107.6 42.4 100.7 26.6 AD-1297643.1 171.8 19.3 163.5 59.8 148.8 39.6144.6 47.4 AD-1297644.1 120.4 27.8 116.4 74.3 159.7 41.0 146.9 35.2AD-1297645.1 132.7 16.5 124.9 68.2 135.4 47.3 131.8 14.5 AD-1136073.2107.0 29.3 84.4 19.5 110.2 40.6 87.2 13.5 AD-1136074.2 64.0 37.4 75.59.9 77.0 43.9 46.7 17.6 AD-1136075.2 57.4 28.4 55.8 14.3 75.8 54.4 105.12.4 AD-1136076.2 134.2 49.0 77.6 41.8 113.1 45.4 129.4 22.5 AD-1136077.2113.0 39.9 120.7 46.9 137.8 37.8 142.3 14.0 AD-1136078.2 131.8 55.1178.5 56.6 162.1 67.7 123.9 5.3 AD-1297646.1 93.7 34.8 136.6 47.1 166.910.2 131.9 14.5 AD-1297647.1 93.6 35.1 64.8 30.2 114.4 17.8 102.2 31.1AD-1297648.1 93.2 37.3 71.0 18.1 105.6 16.3 50.9 6.0 AD-1297649.1 42.19.3 48.7 24.7 42.6 20.2 53.8 11.5 AD-1297650.1 99.4 15.2 152.2 18.6135.9 21.5 121.5 16.7 AD-1297651.1 95.1 26.3 108.1 38.1 85.9 24.5 102.821.6 AD-1297652.1 154.3 23.3 135.2 52.7 139.4 29.3 132.3 10.5AD-1297653.1 83.7 34.5 76.3 32.5 138.2 47.7 140.0 10.9 AD-1297654.1117.1 41.4 71.0 25.4 141.1 68.1 90.2 6.9 AD-1297655.1 86.2 32.0 94.823.8 108.4 27.9 83.0 19.5 AD-1297656.1 82.2 45.6 59.4 28.2 75.7 44.538.7 16.3 AD-1297657.1 26.2 3.2 23.2 11.8 54.0 51.9 55.2 27.3AD-1297658.1 72.2 16.6 82.9 48.2 72.9 51.9 84.7 25.9 AD-1297659.1 64.927.0 60.3 13.8 91.4 40.8 66.0 24.7 AD-1297660.1 63.0 27.9 98.7 51.5 77.769.3 93.3 33.9 AD-1297661.1 39.7 24.5 68.4 30.8 69.3 32.7 93.8 25.5AD-1297662.1 58.6 25.8 42.1 12.7 48.2 30.1 64.3 16.4 AD-1297663.1 50.311.1 24.4 8.9 39.7 19.9 53.4 15.9 AD-1136082.2 16.1 10.8 64.9 27.2 81.520.2 81.5 20.2 AD-1136083.2 51.2 13.6 72.2 16.5 102.0 14.1 102.0 14.1AD-1136084.2 55.5 32.4 98.3 30.3 112.8 22.6 112.8 22.6 AD-1136085.2 57.835.2 84.0 23.8 124.3 4.7 124.3 4.7 AD-1136086.2 79.6 24.1 112.1 8.6 92.629.1 92.6 29.1 AD-1136087.2 62.4 7.5 80.3 19.8 93.5 13.7 93.5 13.7AD-1136088.2 64.3 10.4 96.8 8.3 94.2 30.4 94.2 30.4 AD-1136089.2 40.831.0 55.3 15.4 88.4 23.2 88.4 23.2 AD-1136090.2 61.4 30.3 131.1 32.5137.3 33.0 137.3 33.0 AD-1297664.1 96.5 25.0 100.0 21.1 135.0 35.2 135.035.2 AD-1136091.2 74.3 29.0 84.0 28.1 95.3 11.3 95.3 11.3 AD-1136092.283.0 31.0 66.2 14.4 108.0 7.5 108.0 7.5 AD-1297665.1 62.2 25.2 68.7 20.688.4 9.3 88.4 9.3 AD-1297666.1 51.2 13.0 47.1 7.5 78.9 12.0 78.9 12.0AD-1297667.1 55.4 18.2 67.5 21.1 70.5 10.2 70.5 10.2 AD-1297668.1 50.918.1 70.6 15.2 98.5 19.5 98.5 19.5 AD-1297669.1 52.7 18.5 99.2 21.6103.8 19.6 103.8 19.6 AD-1297670.1 80.5 26.4 88.7 32.0 113.9 8.1 113.98.1 AD-1297671.1 71.4 24.3 71.7 7.8 98.7 7.6 98.7 7.6 AD-1297672.1 103.04.8 97.6 3.4 119.4 33.0 119.4 33.0 AD-1297673.1 47.8 19.5 60.8 21.7 98.410.1 98.4 10.1 AD-1297674.1 96.5 17.6 86.3 14.8 93.4 11.4 93.4 11.4AD-1297675.1 42.9 9.7 72.8 16.2 80.5 10.6 80.5 10.6 AD-1136159.2 76.720.7 65.0 25.8 107.4 22.5 107.4 22.5 AD-1136160.2 74.6 15.1 77.2 20.1131.3 1.6 131.3 1.6 AD-1136161.2 81.1 33.0 80.1 20.6 103.6 26.3 103.626.3 AD-1136162.2 66.4 12.3 88.3 37.3 124.8 13.6 124.8 13.6 AD-1136163.269.6 18.4 50.6 8.3 89.5 11.6 89.5 11.6 AD-1297676.1 55.7 30.9 51.5 14.178.1 17.8 78.1 17.8 AD-1136164.2 41.7 11.4 79.9 27.9 72.8 23.5 72.8 23.5AD-1136165.2 57.4 18.9 91.6 15.6 109.6 20.4 109.6 20.4 AD-1136166.2 70.719.9 62.2 24.4 110.2 28.8 110.2 28.8 AD-1136167.2 117.2 19.8 100.5 32.0145.2 13.9 145.2 13.9 AD-1136168.2 72.9 11.4 69.2 12.1 115.2 25.9 115.225.9 AD-1136169.2 52.4 6.6 39.9 7.6 105.5 15.2 105.5 15.2 AD-1136170.260.2 13.0 58.5 34.9 88.9 22.0 88.9 22.0 AD-1136171.2 93.8 9.6 75.3 11.172.9 15.5 72.9 15.5 AD-1136172.2 55.6 25.3 65.7 17.7 83.9 21.8 83.9 21.8AD-1136173.2 79.0 36.1 120.7 34.2 127.9 14.1 127.9 14.1 AD-1297677.189.9 17.7 100.3 37.6 90.2 34.0 90.2 34.0 AD-1136174.2 84.7 10.4 94.928.6 113.4 16.5 113.4 16.5 AD-1297678.1 110.9 29.0 76.8 13.1 140.0 13.6140.0 13.6 AD-1297679.1 115.4 5.5 120.9 35.8 122.2 23.8 122.2 23.8AD-1297680.1 98.6 13.3 102.9 31.1 103.7 0.8 103.7 0.8 AD-1297681.1 97.114.8 84.5 27.3 95.6 17.1 95.6 17.1 AD-1297682.1 95.1 15.9 111.8 16.7116.9 32.7 116.9 32.7 AD-1297683.1 101.0 20.4 85.3 21.6 128.9 12.9 128.912.9 AD-1297684.1 92.9 24.8 69.1 12.3 109.7 21.6 109.7 21.6 AD-1297685.154.7 12.1 67.4 27.5 103.5 5.8 103.5 5.8 AD-1297686.1 81.4 10.3 98.4 30.3115.9 14.0 115.9 14.0 AD-1297687.1 62.0 8.1 63.0 18.0 102.1 8.7 102.18.7 AD-1297688.1 41.4 12.5 49.4 10.4 74.8 20.3 74.8 20.3 AD-1297689.161.0 16.6 51.0 12.1 58.3 8.2 58.3 8.2 AD-1297690.1 64.4 28.9 89.6 27.5116.6 25.3 116.6 25.3 AD-1297691.1 75.7 22.0 99.7 24.9 123.5 26.7 123.526.7 AD-1297692.1 130.0 27.2 100.0 30.0 127.8 23.3 127.8 23.3AD-1136221.2 86.4 21.5 72.9 42.0 107.5 5.2 107.5 5.2 AD-1136222.2 142.115.6 109.6 38.9 113.9 6.6 113.9 6.6 AD-1297693.1 75.4 15.8 88.4 20.895.6 11.1 95.6 11.1 AD-1297694.1 77.5 15.8 55.3 3.2 76.3 13.9 76.3 13.9AD-1136223.2 64.0 15.5 46.8 13.9 57.7 9.7 57.7 9.7 AD-1136224.2 69.529.1 66.2 20.4 101.5 27.0 101.5 27.0 AD-1136225.2 98.8 35.1 70.1 20.9117.7 25.2 117.7 25.2 AD-1297695.1 69.5 18.8 88.6 37.0 128.0 25.9 128.025.9 AD-1297696.1 61.8 23.2 50.8 18.2 102.3 23.1 102.3 23.1 AD-1297697.195.3 13.1 68.6 5.8 100.1 15.8 100.1 15.8 AD-1136226.2 47.2 12.2 63.1 9.280.5 41.8 80.5 41.8 AD-1136227.2 55.1 8.6 44.5 7.5 64.3 11.1 64.3 11.1AD-1136228.2 84.9 13.5 94.7 27.3 91.2 17.2 91.2 17.2 AD-1136229.2 91.910.5 95.3 27.5 116.4 23.4 116.4 23.4 AD-1136230.2 119.7 22.0 68.4 23.2106.2 9.2 106.2 9.2 AD-1136231.2 85.7 6.8 76.5 31.1 94.5 13.6 94.5 13.6AD-1136232.2 72.4 12.4 67.0 11.6 94.9 23.9 94.9 23.9 AD-1297698.1 61.613.7 54.5 8.9 83.4 26.1 83.4 26.1 AD-1297699.1 46.0 5.9 55.4 14.6 66.614.0 66.6 14.0 AD-1297700.1 92.2 41.6 95.2 10.8 114.1 24.6 114.1 24.6AD-1136233.2 117.7 27.6 117.3 16.1 110.6 8.0 110.6 8.0 AD-1136234.2108.2 22.1 62.1 4.6 87.5 17.3 87.5 17.3 AD-1297701.1 102.5 11.9 84.412.8 74.9 8.8 74.9 8.8 AD-1297702.1 121.5 19.7 76.0 6.6 91.9 21.3 91.921.3 AD-1297703.1 104.9 10.6 67.7 10.4 79.2 10.8 79.2 10.8 AD-1297704.161.2 22.3 75.7 9.8 85.7 9.3 85.7 9.3 AD-1297705.1 68.6 36.2 89.9 32.271.0 7.8 71.0 7.8 AD-1297706.1 49.9 41.2 93.7 9.8 95.2 24.0 95.2 24.0AD-1297707.1 83.3 18.6 107.2 7.7 106.5 8.3 106.5 8.3 AD-1297708.1 95.815.7 93.9 27.1 86.3 10.0 86.3 10.0 AD-1297709.1 89.9 23.6 92.7 20.2 83.514.1 83.5 14.1 AD-1297710.1 74.2 20.8 94.7 10.3 74.2 3.5 74.2 3.5AD-1297711.1 59.4 36.2 114.5 22.5 72.3 3.0 72.3 3.0 AD-1297712.1 87.312.2 88.3 7.6 88.9 10.3 88.9 10.3

TABLE 10 Xanthine Dehydrogenase dsRNA Agent In Vitro Single Dose Screensin Primary Human Hepatocytes Dose (nM) 100 20 4 0.8 0.16 0.032 0.00640.00128 0.000256 5.12E−05 % Message AD-1395794.1 30.7 30.6 40.2 35.936.1 33.5 55.7 53.3 69.1 60.5 Remaining Avg AD-1136038.3 43.5 36.9 40.535.5 43.0 59.8 61.4 61.1 94.7 76.9 AD-1395797.1 45.8 45.8 53.6 68.5 58.970.2 100.1 89.7 128.6 105.1 AD-1135991.3 42.0 44.8 32.0 54.1 54.0 61.768.4 71.8 91.1 104.4 AD-1297597.2 56.7 51.3 46.3 43.4 56.9 76.7 86.589.4 75.7 84.5 AD-1395803.1 39.4 43.6 35.7 42.3 53.2 48.2 82.5 79.5 79.169.3 AD-1395805.1 32.7 45.1 34.5 43.1 43.3 81.0 84.0 76.3 85.7 100.7 PBSavg 106.5 Stdev AD-1395794.1 9 7 8 7 10 2 18 11 19 17 AD-1136038.3 11 118 9 5 10 16 16 19 10 AD-1395797.1 1 10 9 8 18 22 10 14 13 34AD-1135991.3 13 14 15 15 17 10 18 18 22 35 AD-1297597.2 7 8 4 14 9 16 1216 31 8 AD-1395803.1 10 5 4 15 18 17 14 28 16 19 AD-1395805.1 8 16 6 6 927 24 20 15 25

TABLE 11 Xanthine Dehydrogenase dsRNA Agent In Vitro Single Dose Screensin Primary Human Hepatocytes Dose (nM) 100 20 4 0.8 0.16 0.032 0.00640.00128 0.000256 5.12E−05 % Message AD-1395807.1 40.3 36.9 38.3 29.843.8 53.9 67.0 75.2 82.8 94.4 Remaining Avg AD-1395811.1 45.7 42.5 39.947.9 64.4 64.7 80.8 99.3 114.2 98.0 AD-1297663.2 45.6 37.8 36.3 44.248.4 57.6 60.0 73.9 92.3 85.7 AD-1136008.2 46.4 50.6 39.3 42.2 49.6 69.486.4 94.2 120.7 102.0 AD-1395816.1 49.1 56.9 54.4 52.0 47.0 60.6 79.4115.4 96.7 97.6 AD-1136061.2 46.9 52.1 48.3 49.4 45.5 66.8 77.0 103.3129.1 99.5 AD-1135987.2 43.4 35.6 34.5 38.2 35.6 52.6 67.4 84.2 88.289.4 PBS avg 100.3 Stdev AD-1395807.1 12 7 7 5 8 14 10 11 11 13AD-1395811.1 9 9 9 7 9 3 11 21 13 20 AD-1297663.2 5 12 10 12 4 7 8 18 2619 % Message AD-1395807.1 40.3 36.9 38.3 29.8 43.8 53.9 67.0 75.2 82.894.4 Remaining Avg AD-1136008.2 2 12 9 9 7 9 18 24 27 22 AD-1395816.1 61 7 7 5 11 6 29 17 30 AD-1136061.2 4 7 10 6 8 6 24 18 28 13 AD-1135987.24 8 8 6 6 14 23 12 8 23

TABLE 12 Xanthine Dehydrogenase dsRNA Agent In Vitro Single Dose Screensin Primary Human Hepatocytes Dose (nM) 100 20 4 0.8 0.16 0.032 0.00640.00128 0.000256 % Message AD-1395823.1 37.7 47.3 42.1 44.0 48.8 64.974.3 93.8 89.7 Remaining Avg AD-1136166.3 47.2 50.7 42.3 49.4 64.4 77.186.3 98.9 102.1 AD-1136169.3 48.0 49.2 38.7 45.8 61.7 67.0 64.8 81.2111.5 PBS avg 100.5 Stdev AD-1395823.1 11 9 13 10 21 12 11 30 15AD-1136166.3 15 14 14 12 12 11 8 23 14 AD-1136169.3 23 5 13 8 9 18 9 528

TABLE 13 IC₅₀ for Selected Xanthine Dehydrogenase dsRNA AgentsDetermined from In Vitro Single Dose Screens in Primary HumanHepatocytes Duplex ID IC50 (nM) AD-1395794.1 0.005 AD-1136038.3 0.057AD-1395797.1 17.28 AD-1135991.3 0.369 AD-1297597.2 0.338 AD-1395803.10.127 AD-1395805.1 0.345 AD-1395807.1 0.055 AD-1395811.1 0.614AD-1297663.2 0.087 AD-1136008.2 0.311 AD-1395816.1 0.25 AD-1136061.2 0.4AD-1135987.2 0.032 AD-1395823.1 0.184 AD-1136166.3 1.149 AD-1136169.30.435

Example 4. In Vivo Screen in Non-Human Primates

Duplexes of interest identified from the in vitro studies were evaluatedin vivo. In particular, the pharmacodynamic activity of variousGalNAc-conjugated siRNAs targeting XDH was analyzed in Cynomolgusmonkeys following a single subcutaneous injection of the siRNAs.Cynomolgus monkeys (3 females/group) in Groups 1 through 12 weresubcutaneously administered vehicle (1×PBS) or a single 10 mg/kg dose ofAD-1136091, AD-1395794, AD-1395805, AD-1136008, AD-1136038, AD-1395823,AD-1395816, AD-1136061, AD-1395811, or AD-1136166, or a single 5 mg/kgdose of AD-1135991. Group 13 was administered Allopurinol in vehicle of0.5% carboxymethylceliulose sodium salt (prepared with sterile water forinjection) at 10 mg/kg via daily oral gavage administration.

Liver samples were collected from Groups 1, 2, 5, 9, and 12 on Days −15,21, 43 and Day 64. Liver samples were collected from Groups 3, 4, 6, 7,8, 10, and 11 on Days −15, 21, and 42. Liver samples were collected fromGroup 13 on Day −15 and Day 15. All samples were analyzed for the levelsof XDH messenger RNA (RT-qPCR) and XDH protein (Western blot).

For the RT-qPCR analysis, the mean liver XDH mRNA level relative toglycerol-3-phosphate dehydrogenase (GAPDH)] from each individual animalwas normalized to its respective predose sample to determine thepercentage of XDH mRNA relative to predose at each specified time point(FIG. 2 ).

Liver XDH protein levels were analyzed using western blot. The liver XDHprotein level relative to beta-actin for each individual animal wasnormalized to its respective predose level to determine the percentageof XDH protein relative to predose at each specified time point (FIG. 3).

As shown in FIGS. 2 and 3 , the greatest silencing for XDH mRNA wasobserved on Day 21 post dose with a maximal suppression of 66% observedfor the AD-1136091 group. Partial recovery was observed on Days 43 and64. A greater extent of silencing was observed for liver XDH proteinwith maximal suppression observed on Days 21 or 43 depending on theduplex tested. Up to a 91% reduction in XDH liver protein level wasobserved for animals treated with AD-1136091 with partial recovery seenby Day 64 post-dose. These results demonstrate that treatment with theexemplary duplex agents targeting XDH effectively reduced both the mRNAlevel and the protein level of XDH in vivo.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

We claim:
 1. A method of treating a subject having a disorder that wouldbenefit from reduction in xanthine dehydrogenase (XDH) expression, themethod comprising administering to the subject a therapeuticallyeffective amount of a double stranded ribonucleic acid (dsRNA) agentcomprising a sense strand and an antisense strand forming a doublestranded region, wherein the sense strand comprises at least 19contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of nucleotides 2701-2721 of SEQ ID NO: 1, and theantisense strand comprises at least 19 contiguous nucleotides from thecorresponding nucleotide sequence of SEQ ID NO: 2, wherein all of thenucleotides of the sense strand and all of the nucleotides of theantisense strand are modified nucleotides, wherein the sense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus, and wherein the antisense strand comprises twophosphorothioate internucleotide linkages at the 5′-terminus and twophosphorothioate internucleotide linkages at the 3′-terminus. therebytreating the subject having the disorder that would benefit fromreduction in XDH expression.
 2. The method of 1, wherein the disorder isan XDH-associated disease.
 3. The method of claim 2, wherein theXDH-associated disease is hyperuricemia.
 4. The method of claim 2,wherein the XDH-associated disease is gout.
 5. The method of claim 1,wherein the subject is human.
 6. The method of claim 1, wherein thedsRNA agent is administered to the subject at a dose of about 0.01 mg/kgto 50 mg/kg.
 7. The method of claim 1, wherein the dsRNA agent isadministered to the subject subcutaneously.
 8. The method of claim 1,further comprising administering to the subject an additionaltherapeutic agent for the treatment of an XDH-associated disease.
 9. Themethod of claim 1, wherein at least one of the modified nucleotides isselected from the group consisting of a deoxy-nucleotide, a 3′-terminaldeoxythimidine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a lockednucleotide, an unlocked nucleotide, a conformationally restrictednucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide,2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga phosphorothioate group, a nucleotide comprising a methylphosphonategroup, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a5′-phosphate mimic, a nucleotide comprising a 2′-phosphate, a thermallydestabilizing nucleotide, a glycol modified nucleotide (GNA), and a2-O—(N-methylacetamide) modified nucleotide; and combinations thereof.10. The method of claim 1, wherein the sense strand and the antisensestrand are each independently 19-25 nucleotides in length.
 11. Themethod of claim 1, wherein the dsRNA agent further comprises a ligand.12. The method of claim 11, wherein the ligand is conjugated to the 3′end of the sense strand of the dsRNA agent.
 13. The method of claim 12,wherein the ligand is one or more GalNAc derivatives attached through amonovalent, bivalent, or trivalent branched linker.
 14. The method ofclaim 13, wherein the ligand is


15. The method of claim 14, wherein the dsRNA agent is conjugated to theligand as shown in the following schematic

wherein X is O or S.
 16. The method of claim 1, wherein the antisensestrand comprises the nucleotide sequence 5′-ACUCGUUCCAUAAUACUCUGAGA-3′(SEQ ID NO:494).
 17. The method of claim 16, wherein the sense strandcomprises the nucleotide sequence 5′-UCAGAGUAUUAUGGAACGAGU-3′(SEQ IDNO:135) and the antisense strand comprises the nucleotide sequence5′-ACUCGUUCCAUAAUACUCUGAGA-3′(SEQ ID NO:494).
 18. The method of claim17, wherein the sense strand comprises the nucleotide sequence5′-uscsagagUfaUfUfAfuggaacgagu-3′(SEQ ID NO:853) and the antisensestrand comprises the nucleotide sequence5′-asCfsucgUfuccauaaUfaCfucugasgsa-3′(SEQ ID NO:1212), wherein a, g, c,and u are 2′-O-methyl (2′-OMe) A, G, C, and U, respectively; Af, Gf, Cfand Uf are 2′-fluoro A, G, C and U, respectively; and s is aphosphorothioate linkage.
 19. The method of claim 18, wherein the dsRNAagent further comprises a ligand.
 20. The method of claim 19, whereinthe 3′-end of the sense strand is conjugated to a ligand as shown in thefollowing schematic

wherein X is O.
 21. A method of treating a subject having a disorderthat would benefit from reduction in xanthine dehydrogenase (XDH)expression, the method comprising administering to the subject atherapeutically effective amount of a double stranded ribonucleic acid(dsRNA) agent comprising a sense strand and an antisense strand forminga double stranded region, wherein the sense strand comprises thenucleotide sequence 5′-uscsagagUfaUfUfAfuggaacgagu-3′(SEQ ID NO:853) andthe antisense strand comprises the nucleotide sequence5′-asCfsucgUfuccauaaUfaCfucugasgsa-3′(SEQ ID NO:1212), wherein a, g, c,and u are 2′-O-methyl (2′-OMe) A, G, C, and U, respectively; Af, Gf, Cfand Uf are 2′-fluoro A, G, C and U, respectively; and s is aphosphorothioate linkage; and wherein the 3′-end of the sense strand isconjugated to a ligand as shown in the following schematic

wherein X is O, thereby treating the subject having the disorder thatwould benefit from reduction in XDH expression.
 22. The method of claim21, wherein the disorder is an XDH-associated disease.
 23. The method ofclaim 22, wherein the XDH-associated disease is hyperuricemia.
 24. Themethod of claim 22, wherein the XDH-associated disease is gout.
 25. Themethod of claim 21, wherein the subject is human.
 26. The method ofclaim 21, wherein the dsRNA agent is administered to the subject at adose of about 0.01 mg/kg to 50 mg/kg.
 27. The method of claim 21,wherein the dsRNA agent is administered to the subject subcutaneously.28. The method of claim 21, further comprising administering to thesubject an additional therapeutic agent for the treatment of anXDH-associated disease.
 29. The method of claim 28, wherein the sensestrand consists of the nucleotide sequence5′-uscsagagUfaUfUfAfuggaacgagu-3′(SEQ ID NO:853) and the antisensestrand consists of the nucleotide sequence5′-asCfsucgUfuccauaaUfaCfucugasgsa-3′(SEQ ID NO:1212).
 30. The method ofclaim 21, wherein the dsRNA agent is present in a pharmaceuticalcomposition.